Self-assembling multicellular bodies and methods of producing a three-dimensional biological structure using the same

ABSTRACT

Structures and methods for tissue engineering include a multicellular body including a plurality of living cells. A plurality of multicellular bodies can be arranged in a pattern and allowed to fuse to form an engineered tissue. The arrangement can include filler bodies including a biocompatible material that resists migration and ingrowth of cells from the multicellular bodies and that is resistant to adherence of cells to it. Three-dimensional constructs can be assembled by printing or otherwise stacking the multicellular bodies and filler bodies such that there is direct contact between adjoining multicellular bodies, suitably along a contact area that has a substantial length. The direct contact between the multicellular bodies promotes efficient and reliable fusion. The increased contact area between adjoining multicellular bodies also promotes efficient and reliable fusion. Methods of producing multicellular bodies having characteristics that facilitate assembly of the three-dimensional constructs are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/491,228, filed on Jun. 24, 2009, now U.S. Pat. No. 8,143,055, whichclaims the benefit of U.S. Provisional Application Ser. No. 61/132,977,filed on Jun. 24, 2008.

GRANT STATEMENT

The invention was made in part from government support under Grant No.NSF-0526854 from the National Science Foundation. The U.S. Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the field of regenerative medicine andtissue engineering, and more particularly to production of engineeredtissues/organs having desired structures.

BACKGROUND

Tissue engineering provides promising solutions to problems caused bythe growing demand for organ and tissue replacement coupled with achronic shortage of transplantable organs, including blood vessels. Inthe United States, for example, thousands of people are on the nationalwaiting list for organ transplants. Many will likely perish for lack ofreplacement blood vessels for diseased arteries or veins or replacementabdominal organs. To lessen and eventually solve the problem ofinadequate supply of blood vessels and organs for transplantation,tissue engineers strive to build and grow transplantable blood vessels,blood vessel substitutes, organs, or organ substitutes in a laboratory,with high precision, on large scale, and in a relatively short amount oftime.

A variety of methods to build engineered tissues have been attempted anddeveloped with limited success. However, assembly of vascularizedthree-dimensional organs has not been accomplished.

Prior art solutions, though promising, have presented a number ofchallenges. Scaffold choice, immunogenicity, degradation rate, toxicityof degradation products, host inflammatory responses, fibrous tissueformation due to scaffold degradation, and mechanical mismatch with thesurrounding tissue may affect the long term behavior of the engineeredtissue construct and directly interfere with its primary biologicalfunction. For example, myocardial tissue requires high cell density toassure synchronous beating through gap junctions that tightlyinterconnect neighboring cells. The use of scaffolds in cardiac tissueengineering has been associated with reduced cell-to-cell connection, aswell as incorrect deposition and alignment of extracellular matrix (ECM;e.g., collagen and elastin), affecting scaffold biodegradation and theforce-generating ability of myocardial constructs. ECM-related factorsare also particularly critical in vascular tissue engineering. Largelyfor this reason the promise of a scaffold-engineered small-diameterblood vessel substitute with mechanical strength comparable to nativevessels for adult arterial revascularization, often described as the“holy grail” of tissue-engineering, remains unfulfilled. Besides therecurrent difficulty of producing elastic fibers in vitro, the use ofscaffolds presents additional problems. The inherent weakness of thegels may hinder the final strength of the tissue-engineered vessel. Inaddition, the presence of residual polymer fragments can disrupt thenormal organization of the vascular wall and even influence smoothmuscle cell (SMC) phenotype. Therefore it is not surprising that thefirst clinical applications of tissue-engineered vascular grafts haveeither targeted low-pressure applications or relied on an entirelyscaffold-free method termed sheet-based tissue-engineering.

Organ printing, especially the technique described in U.S. patentapplication Ser. No. 10/590,446, has shown promise for producingthree-dimensional tissues. Organ printing is generally a computer-aided,dispenser-based, three-dimensional tissue-engineering technology aimedat constructing functional organ modules and eventually entire organslayer-by-layer. In the technology described in U.S. patent applicationSer. No. 10/590,446, individual multicellular aggregates are printedinto a gel or other support matrix. The final functional tissue resultsform the post-printing fusion of the individual aggregates.

SUMMARY OF THE INVENTION

One aspect of the invention is an elongate multicellular body. The bodyincludes a plurality of living cells and tissue culture medium. Thecells are cohered to one another. The multicellular body has a length ofat least about 1000 microns and an average cross-sectional area alongits length in the range of about 7,850 square microns to about 360,000square microns.

Another aspect of the invention is an engineered elongate multicellularbody. The body includes a plurality of living cells that are cohered toone another. The multicellular body has a length of at least about 1000microns and an average cross-sectional area along its length in therange of about 7,850 square microns to about 360,000 square microns.

Another embodiment is a non-innervated and non-cartilaginous elongatemulticellular body. The body includes a plurality of living cells thatare cohered to one another. The multicellular body has a length of atleast about 1000 microns and an average cross-sectional area along itslength in the range of about 7,850 square microns to about 360,000square microns.

A further aspect of the invention is a lumenless elongate multicellularbody. The body includes a plurality of living cells and tissue culturemedium. The cells are cohered to one another. The multicellular body hasan aspect ratio that is at least about 2.

In one aspect of the invention, a multicellular body made of a pluralityof cells or cell aggregates in a desired three-dimensional shape withviscoelastic consistency is described. The multicellular body comprisesa plurality of cells or cell aggregates, wherein the cells or cellaggregates cohere together to form a construct in a pre-determined shapewith viscoelastic consistency, desired cell density, and sufficientintegrity for easy manipulation and handling.

Yet another aspect of the invention is a method of producing an elongatemulticellular body that includes a plurality of living cells. A cellpaste including a plurality of living cells is shaped into an elongateshape. The shaped cell paste is incubated in a controlled environment toallow the cells to cohere to one another to form the elongatemulticellular body.

Another method of producing a multicellular body including a pluralityof living cells according to the invention includes shaping a cell pastethat includes a plurality of living cells in a device that holds thecell paste in a three-dimensional shape. The shaped cell paste isincubated a controlled environment while it is held in saidthree-dimensional shape for a sufficient time to produce a body that hassufficient cohesion to support itself on a flat surface.

Also provided is a method of producing an elongate multicellular bodycomprising a plurality of living cells. The method comprises shaping acell paste comprising a plurality of living cells into an elongateshape, and incubating the shaped cell paste in a controlled environmentto allow the cells to cohere to one another to form the elongatemulticellular body. In this aspect of the invention, a method forproducing an elongate multicellular body, which comprises a plurality ofcells or cell aggregates in a pre-determined shape with viscoelasticconsistency, is described. In one embodiment, the method to produce amulticellular body comprises the steps of: 1) providing a cell pastecontaining a plurality of pre-selected cells or cell aggregates with adesired cell density and viscosity, 2) manipulating the cell paste intodesired shape, and 3) forming the multicellular body through maturation.

In yet another aspect of the invention, a filler body which is used incombination with the aforesaid multicellular body to build a desiredthree-dimensional biological construct, is described. The filler bodycomprises a material in a pre-determined shape, where the materialresists the in-growth, migration, and adherence of cells, and can alsobe permeable to tissue culture media (i.e., permeable to nutrients). Thefiller body may be made of material such as agarose, agar, and/or otherhydrogels. During the construction of a biological construct, the fillerbodies are employed, according to a pre-determined pattern, to definedomains void of the multicellular bodies.

In another aspect of the invention, a method of forming the fillerbodies is described. In general, the method is to prepare (e.g.,manipulate) a pre-selected suitable material in a gel-like conditioninto a desired shape. According to one embodiment of the inventivemethod, the fabrication method may further include the steps of: 1)lowering the viscosity of the material to liquid-like material, 2)shaping the liquid-like material with a pre-determined shape, and 3)raising the viscosity of the material into that of the desired gel-likefiller matrix unit.

Yet another embodiment of the invention is a three-dimensional structureincluding a plurality of non-innervated elongate multicellular bodies.Each multicellular body includes a plurality of living cells cohered toone another. The multicellular bodies are arranged in a pattern in whicheach multicellular body contacts at least one other multicellular bodyand the multicellular bodies are not cohered to one another.

A further aspect of the invention is a three-dimensional structure. Thestructure includes a plurality of engineered elongate multicellularbodies. Each multicellular body includes a plurality of living cellscohered to one another. The multicellular bodies are arranged in apattern in which each multicellular body contacts at least one othermulticellular body and the multicellular bodies are not cohered to oneanother.

In another embodiment, a three-dimensional structure includes aplurality of elongate multicellular bodies. Each multicellular bodyincludes a plurality of living cells cohered to one another and tissueculture medium. The multicellular bodies are arranged in a pattern inwhich each multicellular body contacts at least one other multicellularbody and the multicellular bodies are not cohered to one another.

In yet another embodiment, a three-dimensional structure includes aplurality of non-innervated multicellular bodies. Each multicellularbody includes a plurality of living cells cohered to one another. Themulticellular bodies are arranged in a pattern in which at least one ofthe multicellular bodies contacts another of the multicellular bodiesalong a contact area having a length that is at least about 1000microns.

In another aspect of the invention, a three-dimensional structureincludes a plurality of engineered multicellular bodies. Eachmulticellular body includes a plurality of living cells cohered to oneanother. The multicellular bodies are arranged in a pattern in which atleast one of the multicellular bodies contacts another of themulticellular bodies along a contact area having a length that is atleast about 1000 microns.

In yet another embodiment of the invention a three-dimensional structureincludes a plurality of multicellular bodies. Each multicellular bodyincludes a plurality of living cells cohered to one another and tissueculture medium. The multicellular bodies are arranged in a pattern inwhich at least one of the multicellular bodies contacts another of themulticellular bodies along a contact area having a length that is atleast about 1000 microns.

Another embodiment of a three-dimensional structure includes a pluralityof multicellular bodies. Each multicellular body includes a plurality ofliving cells cohered to one another. The structure also includes aplurality of discrete filler bodies. Each filler body includes abiocompatible material that resists migration and ingrowth of cells fromthe multicellular bodies into the filler bodies and resists adherence ofcells in the multicellular bodies to the filler bodies. Themulticellular bodies and filler bodies are arranged in a pattern inwhich each multicellular body contacts at least one other multicellularbody or at least one filler body.

Another further aspect of the invention is a three-dimensional structureincluding a plurality of multicellular bodies. Each multicellular bodyincludes a plurality of living cells cohered to one another. Thestructure also includes a plurality of filler bodies. Each filler bodyincludes a biocompatible material that resists migration and ingrowth ofcells from the multicellular bodies into the filler bodies and resistsadherence of cells in the multicellular bodies to the filler bodies. Themulticellular bodies and the filler bodies are arranged to form aplurality of spaces in the three dimensional structure that are notoccupied by the multicellular bodies and that are not occupied by thefiller bodies.

Yet another aspect of the invention is a method of producing athree-dimensional biological engineered tissue. The method includesarranging a plurality of elongate multicellular bodies according to apattern such that each of the multicellular bodies contacts at least oneother multicellular body. Each multicellular body includes a pluralityof living cells. At least one of the multicellular bodies is allowed tofuse with at least one other multicellular body.

In another embodiment of a method of producing a three-dimensionalbiological engineered tissue, a plurality of multicellular bodies and aplurality of filler bodies are arranged according to a pattern such thateach of the multicellular bodies contacts at least one of (i) anothermulticellular body or (ii) a filler body. Each multicellular bodyincludes a plurality of living cells. Each filler body includes abiocompatible material that resists migration and ingrowth of cells fromthe multicellular bodies into the biocompatible material and resistsadherence of cells in the multicellular bodies to the filler bodies. Atleast one of the multicellular bodies is allowed to fuse with at leastone other multicellular body.

In yet another embodiment of a method of producing a three-dimensionalbiological engineered tissue, the method is to deliver a plurality ofmulticellular bodies into a pre-determined pattern in a pre-selectedreceiving environment. According to one embodiment of the engineeringmethod, the multicellular bodies may be employed in combination with thepre-selected filler bodies. More particularly, in one embodiment, themethod includes the steps of: 1) delivering the plurality ofmulticellular bodies in a pre-determined combination with a plurality offiller bodies according to the pre-determined pattern to form a stackedor layered construct, where the multicellular bodies and the fillerbodies are contiguous, 2) depositing the layered construct into apre-selected controlled environment for maturation, whereby themulticellular bodies fuse with each other to result in a fusedconstruct, and 3) removing the filler bodies from the fused construct toproduce the desired biological construct.

Another embodiment of the invention is a three-dimensional structureincluding at least one filler body and a plurality of living cells whichare cohered to one another. The cells form a tubular structuresubstantially surrounding the at least one filler body. The filler bodyincludes a compliant biocompatible material that resists migration andingrowth of cells into the material and which resists adherence of cellsto the material.

Still another aspect of the invention is a mold for producing amulticellular body comprising a plurality of living cells cohered to oneanother. The mold has biocompatible substrate that is resistant tomigration and ingrowth of cells into the substrate and resistant toadherence of cells to the substrate. The substrate is shaped to receivea composition comprising plurality of cells having a relatively lowercohesion and hold the composition in a desired shape during a maturationperiod during which the cohesion increases to form the multicellularbody. The desired shape of the multicellular body has a length of atleast about 1000 microns and is configured so every cell within themulticellular body is no more than about 250 microns from an exterior ofthe body.

Another embodiment of the invention is a tool for making a mold that issuitable for producing a plurality of multicellular bodies in which eachbody includes a plurality of living cells cohered to one another. Thetool has a body having a top and a bottom. A plurality of fins extendfrom the bottom of the body. Each of the fins has a width in the rangeof about 100 microns to about 800 microns for forming grooves in abiocompatible gel substrate configured for forming living cells placedin the grooves into elongate multicellular bodies. The fins havelongitudinal axes and at least one of the fins is spaced laterally fromthe longitudinal axis of another of the fins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a perspective of one embodiment of a multicellular body ofthe present invention;

FIG. 1B is an enlarged perspective of the multicellular body supportedby a surface;

FIG. 1C is a an enlarged perspective of the ends of multiplemulticellular bodies in side-by-side adjoining relation to one anotheron the surface;

FIG. 2 is a perspective of one embodiment of a three-dimensionalconstruct including a plurality of the multicellular bodies and aplurality of filler bodies arranged in a pattern suitable for producingone embodiment of an engineered tissue;

FIGS. 3A-3D illustrate one embodiment of a method of making themulticellular bodies illustrated in FIGS. 1A, 1B, 1C, and 2;

FIG. 4A is a perspective of one embodiment of a mold that is suitablefor use in the method illustrated in FIGS. 3A-3D;

FIG. 4B is a top plan view of the mold;

FIG. 4C is a cross section of the mold taken in a plane including line4C-4C on FIG. 4B;

FIG. 4D is an enlarged cross-section of a portion of the mold asillustrated in FIG. 4C;

FIG. 5A is a perspective of one embodiment of a tool that can be used tomake the mold illustrated in FIGS. 4A-4D;

FIG. 5B is a side view of the tool illustrated in FIG. 5A;

FIG. 5C is an enlarged side view of a portion of the tool as illustratedin FIG. 5B;

FIGS. 6A-6C illustrate one embodiment of a method of using the toolillustrated in FIGS. 5A-5C to make the mold illustrated in FIGS. 4A-4D;

FIGS. 7, 7A, and 8-10 are schematic perspectives of various embodimentsof three-dimensional constructs made from a plurality of multicellularbodies and a plurality of filler bodies;

FIGS. 11 and 12 are schematic illustrations of various methods of makingthree-dimensional constructs from a plurality of multicellular bodiesand filler bodies;

FIG. 13 is a photograph of two tubular structures engineered accordingto the methods described herein having outside diameters of 1200 micronsand 900 microns, respectively;

FIG. 14 is a photograph of another tubular structure engineeredaccording to the methods described herein;

FIG. 15 is a schematic perspective of one embodiment of a tubularstructure engineered according to the methods described herein incombination with a filler body in the lumen of the tubular structure;

FIG. 16 includes a group of photographs illustrating fusion of sphericalmulticellular bodies to form a branched tubular structure;

FIG. 17 includes a group of photographs illustrating fusion of a firstset of multicellular bodies with a second set of multicellular bodiesthat have a cell type composition that is different from the compositionof the multicellular bodies in the first set; and

FIG. 18 includes a group of photographs illustrating a tubularengineered structure that includes gelatin and fibrin.

Corresponding reference characters indicate corresponding partsthroughout the drawings.

DETAILED DESCRIPTION

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

New structures and methods for producing engineered tissue are provided.The technology involves use of novel multicellular bodies as buildingblocks that can be used to assemble a three-dimensional construct thatcan become a desired engineered tissue through maturation. Eachmulticellular body comprises a plurality of living cells that aresufficiently cohered to one another to allow the body to be handled(e.g., picked up and moved) as a single object. The cohesion of themulticellular body is suitably sufficient to allow the body to supportitself (e.g., on a work surface or in an assembly that includes multiplemulticellular bodies) for a period of time sufficient to enable theliving cells to cohere to the living cells of an adjoining multicellularbody. The ability to pick up and move a plurality of living cells in theform of a self-supporting multicellular body provides flexibility toassemble numerous different three-dimensional constructs. For example,the multicellular bodies can be used in conjunction with one or morefiller bodies (e.g., bodies comprising a biocompatible material thatresists migration and ingrowth of cells from the multicellular bodiesinto the filler bodies and resists adherence of cells to the fillerbodies) to assemble constructs that can become a tubular engineeredtissue through maturation. The multicellular bodies and filler bodiescan also be used to assemble constructs that become engineered tissueshaving other shapes through maturation. Further, because themulticellular bodies are self-supporting, there is no need to embed themulticellular bodies in a supporting gel or scaffold. Instead, theability to “print in air” facilitates arranging the multicellular bodiesin a manner that ensures the multicellular bodies are in direct contactwith one another. Better contact between the multicellular bodies canfacilitate efficient and reliable fusion of the multicellular bodiesduring maturation. In addition, the filler bodies can be easily removedfrom the exterior and interior (e.g. the lumen of a tubular structure)of a mature engineered tissue.

In addition, some of the methods of the present invention use elongatemulticellular bodies as the building blocks for the engineered tissue.Because elongate multicellular bodies are already cohered to one anotherover a significant length along a longitudinal axis of the body, fusionof the multicellular bodies is more reliable and can be achieved in lesstime. Further, elongate multicellular bodies can be arranged inside-by-side adjoining relation to establish contact between themulticellular bodies along a contact area having a substantial length.This can facilitate rapid and reliable fusion of the adjoiningmulticellular bodies to one another.

Having provided a general overview of a method of producing athree-dimensional biological engineered tissue using the materials andprocesses of the present invention, such processes and materials willnow be described in more detail.

Multicellular Bodies

One embodiment of a multicellular body (also referred to herein as anintermediate cellular unit), generally designated 1, is illustrated inFIG. 1. The multicellular body 1 includes a plurality of living cellsthat are cohered to one another. The multicellular body 1 comprises aplurality of cells cohered together in a desired three-dimensional (3-D)shape with viscoelastic consistency and sufficient integrity for easymanipulation and handling during a bio-engineering process, such astissue or organ engineering. Sufficient integrity means that themulticellular body, during the subsequent handling, is capable ofretaining its physical shape, which is not rigid, but has a viscoelasticconsistency, and maintaining the vitality of the cells.

The multicellular body 1 may be composed of any one or more pre-selectedcell types. In general, the choice of cell type will vary depending onthe desired three-dimensional biological tissue. For example, if themulticellular body is to be used to engineer a blood vessel-typethree-dimensional structure, the cells used to form the multicellularbodies can advantageously comprise a cell type or cell types typicallyfound in vascular tissue (e.g., endothelial cells, smooth muscle cells,fibroblasts, etc.). Other cell types may be used to form themulticellular body if it is to be used to engineer a different type ofthree-dimensional tissue (e.g., intestine, liver, kidney, etc.). Oneskilled in the art will be able to choose an appropriate cell type ortypes for the multicellular body based on the type of three-dimensionaltissue to be engineered. Non-limiting examples of suitable cell typesinclude contractile or muscle cells (e.g., striated muscle cells,including myoblasts and cardiomyocytes, and smooth muscle cells), neuralcells, fibroblasts, connective tissue cells (including the cell typeswhich make up bone and cartilage, cells capable of differentiating intobone forming cells and chondrocytes, and cell types which make up lymphtissues), parenchymal cells, epithelial cells (including endothelialcells that form linings in cavities and vessels or channels, exocrineand endocrine secretory epithelial cells, epithelial absorptive cells,keratinizing epithelial cells, and extracellular matrix secretioncells), hepatocytes, and undifferentiated cells (such as embryoniccells, stem cells, and other precursor cells), among others. Forexample, the cells used to form the multicellular body 1 can be obtainedfrom a live human or animal subject and cultured as a primary cell line.

The multicellular body 1 may be homocellular or heterocellular. Inhomocellular multicellular bodies, the plurality of living cellsincludes a plurality of living cells of a single cell type. Almost allof the living cells in a homocellular multicellular body are cells ofthe single cell type, subject to some tolerance for low levels ofimpurities including a relatively small number of cells of a differentcell type that have no more than a negligible impact on the maturationof a construct including the homocellular multicellular body.

In contrast, a heterocellular multicellular body includes significantnumbers of cells of more than one cell type. For example, amulticellular body can comprise a plurality of living cells of a firsttype and a plurality of living cells of a second type (etc.), the secondcell type being different from the first cell type. If the multicellularbodies are to be used to create vascular tissue, for instance, the cellsof the first type can be endothelial cells and the cells of the secondtype can be smooth muscle cells, the cells of the first type can beendothelial cells and the cells of the second type can be fibroblasts,or the cells of the first type can be smooth muscle cells and the cellsof the second type can be fibroblasts. Heterocellular multicellularbodies can also include a plurality of cells of a first cell type, aplurality of cells of a second cell type, and a plurality of cells of athird cell type with each of the first, second and third cell typesbeing different from the others of the first, second, and third cellstypes. For example, a multicellular body that is suitable for producingan engineered blood vessel can include endothelial cells, smooth musclecells, and fibroblasts. The living cells in a heterocellular body mayremain unsorted or can “sort out” (e.g., self-assemble) during thefusion process to form a particular internal structure for theengineered tissue. The self sorting of cells is consistent with thepredictions of the Differential Adhesion Hypothesis (DAH). The DAHexplains the liquid-like behavior of cell populations in terms of tissuesurface and interfacial tensions generated by adhesive and cohesiveinteractions between the component cells. In general, cells can sortbased on differences in the adhesive strengths of the cells. Forexample, cell types that sort to the center of a heterocellularmulticellular body generally have a stronger adhesion strength (and thushigher surface tension) than cells that sort to the outside of themulticellular body.

Furthermore, when a heterocellular multicellular body is composed ofcells from tissues that are neighbors in normal development, in thecourse of sorting they may recover their physiological conformation.Thus, heterocellular multicellular bodies may comprise a sort ofpre-built internal structure, based on the adhesive and cohesiveproperties of the component cells, and the environment in which thecells are located. This can be used to build more complex biologicalstructures. For example, while building a simple contractile tube,homocellular multicellular bodies composed of muscle cells can be used;to build a blood vessel-like structure, at least two cell types can beused. For example, a heterocellular multicellular body to be used forbuilding an engineered blood vessel can suitably include (i) endothelialcells and smooth muscle cells; (ii) smooth muscle cells and fibroblasts;(iii) endothelial cells and fibroblasts; or (iv) endothelial cells,smooth muscle cells, and fibroblasts. By using multicellular bodiescomposed of these multiple different cell types randomly dispersed inthe body to build a three-dimensional biological structure, in thecourse of structure formation the different cell types can sort out soendothelial cells line the internal structure of the tube (i.e., thelumen), smooth muscle cells form a layer surrounding the endothelialcells, and the fibroblasts form an outer layer surrounding the smoothmuscle layer. The optimal structure can be achieved by varying thecomposition of the multicellular body (e.g., ratios of the variousdifferent cell types to one another) and by size of the multicellularbody. As another example, heterocellular multicellular bodies caninclude a plurality of living cells of a first cell type, a plurality ofcells of a second type, and a plurality of cells of a third type. Ifsuch multicellular bodies are to be used to create vascular tissue, forinstance, the cells of the first cell type can suitably be endothelialcells, the cells of the second cell type can suitably be smooth musclecells, and the cells of the third cell type can suitably be fibroblasts.Again, self-sorting of the cells may occur in such heterocellularmulticellular bodies. Thus, when these multicellular bodies are used tobuild a three-dimensional biological structure, for example a tubularstructure, in the course of structure formation these cell types maysort such that the endothelial cells line the internal structure of thetube (i.e., the lumen), the smooth muscle cells form a layersubstantially surrounding the endothelial cells, and the fibroblastsform the outer layer of the tubular structure, substantially surroundingboth the layer of endothelial cells and the layer of smooth musclecells.

In some instances, the multicellular body 1 suitably includes one ormore extracellular matrix (ECM) components or one or more derivatives ofone or more ECM components in addition to the plurality of cells. Forexample, the multicellular bodies may contain various ECM proteins(e.g., gelatin, fibrinogen, fibrin, collagen, fibronectin, laminin,elastin, and/or proteoglycans). The ECM components or derivatives of ECMcomponents can be added to a cell paste used to form the multicellularbody, as discussed in further detail below. The ECM components orderivatives of ECM components added to the cell paste can be purifiedfrom a human or animal source, or produced by recombinant methods knownin the art. Alternatively, the ECM components or derivatives of ECMcomponents can be naturally secreted by the cells in the multicellularbody, or the cells used to make the multicellular body can begenetically manipulated by any suitable method known in the art to varythe expression level of one or more ECM components or derivatives of ECMcomponents and/or one or more cell adhesion molecules or cell-substrateadhesion molecules (e.g., selectins, integrins, immunoglobulins, andcadherins). The ECM components or derivatives of ECM components maypromote cohesion of the cells in the multicellular body. For example,gelatin and/or fibrinogen can suitably be added to the cell paste whichis used to form the multicellular body. The fibrinogen can then beconverted to fibrin by the addition of thrombin.

As noted above, the multicellular body 1 in some instances suitablyincludes a tissue culture medium. The tissue culture medium can be anyphysiologically compatible medium and will typically be chosen accordingto the cell type(s) involved as is well known in the art. The tissueculture medium may comprise, for example, basic nutrients such as sugarsand amino acids, growth factors, antibiotics (to minimizecontamination), etc.

The cohesion of the cells in the multicellular body 1 is suitablysufficiently strong to allow the multicellular body to retain athree-dimensional shape while supporting itself on a flat surface. InFIG. 1B, for instance, the multicellular body 1 is supporting itself ona flat surface 13. Although there is some minor deformation (e.g., wherethe multicellular body 1 contacts the surface 13), the multicellularbody is sufficiently cohesive to retain a height that is suitably atleast one half its width, and more suitably about equal to the width.Also as illustrated in FIG. 1B, for example, the multicellular body 1 issupported by a flat exterior surface 15 formed on the bottom of themulticellular body by contact between the multicellular body and thesurface 13. When the full weight of the multicellular body 1 issupported by the surface 13, the area A1 of the contact surface 15 maybe larger than the initial contact area due to slight deformation of themulticellular body. However, the area A1 of the contact surface 15 issuitably smaller than the area A2 of a two dimensional projection of themulticellular body 1 onto the support surface 13 (See FIG. 1B). Thismeans that a portion of the multicellular body 1 (e.g., each of thesides as illustrated in FIG. 1B) is supported by the multicellular body1 above the work surface 13. Likewise, when two or more of themulticellular bodies 1 are placed in side-by-side adjoining relation toone another on the flat surface 13 (FIG. 1C), their self-supportingabilities in combination with the three-dimensional shape in which theyretain themselves can form a void space 17 under their sides and abovethe work surface.

The cohesion of the cells in the multicellular body 1 is also suitablysufficiently strong to allow the multicellular body to support theweight of at least one similarly sized and shaped multicellular body orfiller body when the multicellular body is assembled in a construct inwhich the multicellular bodies and filler bodies are stacked on top ofone another (See FIG. 2, discussed in more detail below). The cohesionof the cells in the multicellular body 1 is also suitably sufficientlystrong to allow the multicellular body to be picked up by an implement(e.g., a capillary micropipette) (See FIG. 3D, discussed in more detailbelow).

Furthermore, the multicellular body 1 can suitably be non-innervated(i.e., it is substantially free of neurons) or non-cartilaginous, orboth non-innervated and non-cartilaginous. The multicellular body can bedescribed as an “engineered” multicellular body because it is differentfrom biological structures that arise without the guidance of humaningenuity. In other words, the multicellular body is synthetic, ornon-naturally occurring.

The multicellular body 1 can have various sizes and shapes within thescope of the invention. For example, the multicellular body 1illustrated in FIG. 1 is a lumenless body, meaning that there is no openpassage extending through the multicellular body. For example, themulticellular body 1 suitably has substantially no voids, hollow spacesor the like within the body. This is one difference between themulticellular body 1 illustrated in FIG. 1 and prior art engineeredblood vessels and other prior art tubular engineered tissues.

The multicellular body 1 illustrated in FIG. 1A-1B is configured tolimit cell necrosis caused by inability of oxygen and/or nutrients todiffuse into central portions of the multicellular body. For example,the multicellular body 1 is suitably configured so none of the livingcells therein is more than about 250 microns from an exterior surface ofthe multicellular body, and more suitably so none of the living cellstherein is more than about 200 microns from an exterior of themulticellular body. Because of the proximity of the cells in the centralportions of the multicellular body 1 to the exterior surface of themulticellular body, cells in the multicellular body can be supplied withoxygen and/or nutrients by diffusion thereof from a void space at theexterior surface of the multicellular body toward the central portionsof the body. Although there may be some necrosis of cells in one or moreportions of the multicellular body (e.g., the central portion), thenecrosis is limited.

The multicellular body 1 in FIG. 1 is also an elongate body having alength L1 that is significantly larger than its height H1 and its widthW1. The length L1 of the multicellular body 1 is suitably at least about1000 microns (e.g., in the range of about 1000 microns to about 30centimeters), more suitably at least about 1 centimeter (e.g., in therange of about 1 centimeter to about 30 centimeters), still moresuitably at least about 5 centimeters (e.g., in the range of about 5centimeters to about 30 centimeters). There is no theoretical upperlimit on the length L1 of the multicellular body. Thus, it is recognizedthat it is possible to make a multicellular body having a length inexcess of 30 centimeters (or any arbitrary length different from 30centimeters) within the scope of the invention as long as a person iswilling to overcome practical difficulties associated with making a longmulticellular body, such as obtaining a sufficient quantity of livingcells or handling a long multicellular body etc.

The height H1 and width W1 of the elongate multicellular body 1illustrated in FIG. 1 are suitably significantly less than its lengthL1. For example, the length L1 is suitably at least twice the width W1and at least twice the height H1, meaning the body 1 has an aspect ratio(i.e., the ratio of the length to the longest dimension orthogonal tothe length) that is at least about 2, more suitably at least about 10and still more suitably at least 20. It will be noted from thedescription of the dimensions of the multicellular body 1 above that theaspect ratio can also be considerably higher than 20 within the scope ofthe invention; for example the aspect ratio can be 2000.

The multicellular body 1 illustrated in FIG. 1 also has a relativelynarrow width W1 and a relatively short height H1. For example, theaverage cross-sectional area of the multicellular body 1 along itslength L is suitably in the range of about 7,850 square microns to about360,000 square microns, more suitably in the range of about 31,400square microns to about 250,000 square microns, and still more suitablyin the range of about 31,400 square microns to about 90,000 squaremicrons. For another example, the multicellular body 1 illustrated inFIG. 1 (which is substantially cylindrical and has a circular crosssection) suitably has an average diameter along its length in the rangeof about 100 microns to about 600 microns (corresponding to an averagecross-sectional area in the range of about 7,850 square microns to about282,600 square microns), more suitably in the range of about 200 micronsto about 500 microns (corresponding to an average cross-sectional areain the range of about 31,400 square microns to about 196,250 squaremicrons), and still more suitably in the range of about 200 microns toabout 300 microns (corresponding to an average cross-sectional area inthe range of about 31,400 square microns to about 70,650 squaremicrons).

Although the multicellular body 1 illustrated in FIG. 1 is substantiallycylindrical and has a substantially circular cross section,multicellular bodies having different sizes and shapes are within thescope of the invention. For example, the multicellular body can be anelongate shape (e.g., a cylindrical shape) with a square, rectangular,triangular, or other non-circular cross sectional shape within the scopeof the invention. Likewise, the multicellular body can have a generallyspherical shape, a non-elongate cylindrical shape, or a cuboidal shapewithin the scope of the invention.

Method of Making the Multicellular Bodies

There are various ways to make multicellular bodies having thecharacteristics described above within the scope of the invention. Forexample, a multicellular body can be fabricated from a cell pastecontaining a plurality of living cells or with a desired cell densityand viscosity. The cell paste can be shaped into a desired shape and amulticellular body formed through maturation (e.g., incubation). Inanother example, an elongate multicellular body is produced by shaping acell paste including a plurality of living cells into an elongate shape.The cell paste is incubated in a controlled environment to allow thecells to cohere to one another to form the elongate multicellular body.It yet another example, a multicellular body is produced by shaping acell paste including a plurality of living cells in a device that holdsthe cell paste in a three-dimensional shape. The cell paste is incubatedin a controlled environment while it is held in the three dimensionalshape for a sufficient time to produce a body that has sufficientcohesion to support itself on a flat surface, as described above.

The cell paste can suitably be provided by: (A) mixing the cells or cellaggregates (also referred to herein as “pre-selected” cells or cellaggregates) (may be one or more cell types) and a cell culture medium(also referred to herein as a “pre-selected” medium) (e.g., in apre-determined ratio) to result in a cell suspension (also referred toherein as a cellular mixture), and (B) compacting the cellular mixtureto produce the cell paste with a desired cell density and viscosity. Thecompacting may be achieved by a number of methods, such as byconcentrating a particular cell suspension that resulted from cellculture to achieve the desired cell concentration (density), viscosity,and consistency required for the cell paste. For example, a relativelydilute cell suspension from cell culture may be centrifuged for adetermined time to achieve a cell concentration in the pellet thatallows shaping in a mold. Tangential flow filtration (“TFF”) is anothersuitable method of concentrating or compacting the cells. Compounds mayalso be combined with the cell suspension to lend the extrusionproperties required. Some examples of suitable compounds that may beused in the present invention include collagen, hydrogels, Matrigel,nanofibers, self-assembling nanofibers, gelatin, fibrinogen, etc.

Thus, the cell paste used in these methods is suitably produced bymixing a plurality of living cells with a tissue culture medium, andcompacting the living cells (e.g., by centrifugation). If one or moreECM components, or one or more derivatives of one or more ECM componentsare to be included in the cell paste (as discussed in further detailbelow), the cell pellet can suitably be resuspended in one or morephysiologically acceptable buffers containing the ECM component(s) orderivative(s) of ECM component(s) and the resulting cell suspensioncentrifuged again to form the cell paste.

The cell density of the cell paste desired for further processing mayvary with cell types. The interactions between cells determine theproperties of the cell paste, and different cell types will have adifferent relationship between cell density and cell-cell interaction.The cells may be pre-treated to increase cellular interactions beforeshaping the cell paste. For example, cells may be incubated inside acentrifuge tube after centrifugation in order to enhance cell-cellinteractions prior to shaping the cell paste.

Various methods may be used to shape the cell paste under the presentinvention. For example the cell paste can be manipulated, manuallymolded or pressed (e.g., after concentration/compaction) to achieve thedesired shape. For example, the cell paste may be taken up (e.g.,aspirated) into a preformed instrument, such as a micropipette (e.g., acapillary pipette), that shapes the cell paste to conform to an interiorsurface of the instrument. The cross sectional shape of the micropipette(e.g., capillary pipette) can be circular, square, rectangular,triangular, or other non-circular cross sectional shape. The cell pastemay also be shaped by depositing it into a preformed mold, such as aplastic mold, metal mold, or a gel mold. Furthermore, centrifugalcasting or continuous casting may be used to shape the cell paste.

In one example of the method, the shaping includes retaining the cellpaste in a shaping device to allow the cells to partially cohere to oneanother in the shaping device. For example, as illustrated in FIG. 3A,cell paste 55 can be aspirated into a shaping device 51 (e.g., acapillary pipette) and held in the shaping device for a maturationperiod (also referred to herein as an incubation period) (FIG. 3B) toallow the cells to at least partially cohere to one another. If thecells are able to achieve sufficient cohesion in the first shapingdevice 51, the multicellular body 1 can be produced in a process thathas only a single maturation step (e.g., a single incubation step). Forexample, the method suitably includes shaping the cell paste 55 in asingle shaping device 51 and incubating the shaped cell paste in asingle controlled environment to allow the cells to cohere to oneanother to form the multicellular body 1. If this is the case, theshaping device 51 (e.g., capillary pipette) can suitably be part of aprinting head of a bioprinter or similar apparatus operable toautomatically place the multicellular body in a three-dimensionalconstruct, as will be described in more detail below The inclusion ofECM components or derivatives of ECM components, for example gelatinand/or fibrinogen, in the cell paste may facilitate production of amulticellular body in a single maturation step because such componentscan promote the overall cohesivity of the multicellular body. However,there is a limit to the amount of time cells can remain in a shapingdevice such as a capillary pipette, which provides the cells onlylimited access at best to oxygen and/or nutrients, before viability ofthe cells is impacted.

If the cells cannot be retained in the shaping device 51 for amaturation period long enough to achieve the desired cohesion, thepartially cohered cell paste 55 is suitably transferred from the shapingdevice (e.g., capillary pipette) to a second shaping device 301 (e.g., amold) that allows nutrients and/or oxygen to be supplied to the cellswhile they are retained in the second shaping device for an additionalmaturation period. One example of a suitable shaping device 301 thatallows the cells to be supplied with nutrients and oxygen is illustratedin FIGS. 4A-4D. This shaping device is a mold 301 for producing aplurality of multicellular bodies (e.g., substantially identicalmulticellular bodies). The mold 301 includes a biocompatible substrate303 made of a material that is resistant to migration and ingrowth ofcells into the substrate and resistant to adherence of cells to thesubstrate. The mold 301 may be made of any material that will excludethe cells from growing or migrating into or adhering to the mold. Forexample, the substrate 303 can suitably be made of Teflon® (PTFE),stainless steel, hyaluronic acid, agarose, agarose, polyethylene glycol,glass, metal, plastic, or gel materials (e.g., agarose gel or otherhydrogel), and similar materials.

The substrate 303 is shaped to receive a composition comprisingplurality of cells having a relatively lower cohesion (e.g., from thefirst shaping device 51) and hold the composition in a desiredthree-dimensional shape during a maturation period during which thecohesion of the cells increases to form a multicellular body that has agreater cohesion relative to the composition before the maturationperiod, such as a multicellular body having any of the characteristicsof the multicellular body 1 described above. The mold 301 is alsosuitably configured so tissue culture media can be supplied to the cellpaste 55 (e.g., by dispensing tissue culture media onto the top of themold). For example, as illustrated in FIGS. 4A-4D a plurality ofelongate grooves 305 are formed in the substrate 303. As illustrated inFIG. 4D, the depth D2 of each groove is suitably in the range of about500 microns to about 1000 microns. The bottom of each groove 305 in theillustrated embodiment suitably has an arcuate (e.g., semicircular)cross-sectional shape for forming elongate cylindrical multicellularbodies that have a substantially circular cross-sectional shape. Thewidth W5 of the grooves 305 is suitably slightly larger than the widthof the multicellular body to be produced in the mold 301. For example,the width W5 of the grooves is suitably in the range of about 300microns to about 1000 microns. The spacing between the grooves 305 isnot critical, but it will generally be desirable to space the groovesrelatively close to one another to increase the number of multicellularbodies that can be produced in the mold 301. In the illustratedembodiment for example, the strips of the substrate 303 between thegrooves 305 each have a width W4 that is about 2 mm.

There are various ways to make a suitable mold within the scope of theinvention. For example, FIGS. 5A-5C illustrate one embodiment of a tool,generally designated 201, that can be used to make a mold that issuitable for making the multicellular bodies described above. Ingeneral, a portion of the tool 201 is configured to be a negative of theportion of the mold 301 that retains the partially cohered cell pasteduring the second maturation period. For example, the tool 201 suitablyincludes a body 203 and a plurality of projections 205 extending fromthe body. Each projection 205 is suitably sized and shaped to form adepression or receiving area in the mold substrate that will retain cellpaste 55 in a shape such that none of the cells in thedepression/receiving area formed in the mold by the projection is morethan about 300 microns from an exterior surface of the shaped cellpaste.

The particular tool 201 illustrated in FIGS. 5A-5C is configured toproduce the mold 301 illustrated in FIGS. 4A-4B. The projections 205 areconfigured as a plurality of fins extending from a bottom 207 of thebody 203. Each of the fins 205 is a negative of a one of the grooves 305in the mold 301. The fins 205 have longitudinal axes 209 (FIG. 5A) andare configured to make a mold that can be used to make the elongatemulticellular bodies 1 described above. At least one of the fins 205 isspaced laterally from the longitudinal axis 209 of another of the fins.This is one difference between the tool 201 and a conventional comb thatis used to form wells in a gel for performing gel electophoresis. In theillustrated embodiment, all of the fins 205 are substantially parallelto one another and each fin is spaced laterally from the other finsrelative to their longitudinal axes 209. The fins 205 are suitably allsubstantially identical to one another. Referring to FIG. 5C, each fin205 suitably extends from the body 203 a distance D1 of about 1.5 mm.The distal end of the fins 205 have an arcuate (e.g., semicircular)cross-sectional shape corresponding to the shape of the bottom of thegrooves 305 in the mold 301. The width W3 of each fin is suitably about300 microns to about 1000 microns. The distance W2 separating the finsis suitably about 2 mm. A lip 211 on the tool 201 is suitably configuredto sit on the rim of a cell culture dish to hold the projections abovethe bottom of the dish. The tool 201 can be made of various materialsfrom which the mold is easily separated, such as Teflon® (PTFE),stainless steel, and the like.

To make the mold 301 a cell culture dish 221 is suitably filled with aliquid 223 that can be made to solidify or set up as a gel, asillustrated in FIG. 6A. For example, the liquid can be an agarosesolution 223. The tool 201 is placed on top of the cell culture dish 221(FIG. 6B) so the lip 211 sits on the rim 225 of the cell culture dishand the projections 205 (e.g., fins) extend from the bottom 207 of thetool 201 into the liquid 223. The liquid 223 is allowed to set up toform a solid or gel substrate surrounding the distal ends of theprojections 205 (e.g., fins). Then tool 201 is lifted off the cellculture dish to separate the tool 201 from the newly produced mold 301(FIG. 6C).

Thus, if a second shaping device is used, the partially cohered cellpaste 55 is suitably transferred from the first shaping device 51 (e.g.,a capillary pipette) to the second shaping device (e.g., the mold 301illustrated in FIGS. 4A-4D). The partially cohered cell paste 55 can betransferred by the first shaping device 51 (e.g., the capillary pipette)into the grooves 305 of the mold 301, as illustrated in FIG. 3C. Thus,the method includes transferring the partially cohered cell paste 55 toa second shaping device 301, and retaining the partially cohered cellpaste in the second shaping device to form the multicellular body 1.Following a maturation period in which the mold 301 is incubated alongwith the cell paste 55 retained therein in a controlled environment toallow the cells in the cell paste to further cohere to one another toform the multicellular body 1, the cohesion of the cells will besufficiently strong to allow the resulting multicellular body 1 to bepicked up with an implement 51′, e.g., a capillary pipette asillustrated in FIG. 3D. The capillary pipette 51′ (now containing themature multicellular body 1 that has been picked up out of a groove 305in the mold 301) can suitably be part of a printing head of a bioprinteror similar apparatus operable to automatically place the multicellularbody into a three-dimensional construct, as will be described in moredetail below.

Thus, in one example of the method of making a multicellular bodies 1,the shaping includes retaining the cell paste 55 in a first shapingdevice 51 to allow the cells to partially cohere to one another in thefirst shaping device, transferring the partially cohered cell paste to asecond shaping device 301, and retaining the partially cohered cellpaste in the second shaping device to form the multicellular body 1.However, in some embodiments, such as when gelatin and/or fibrinogen areadded to the cell paste, the cells may sufficiently cohere to form themulticellular body in the first shaping device 51, and the step oftransferring the cell paste 55 to a second shaping device 301 andretaining the cell paste in the second shaping device may beunnecessary.

The first shaping device 51 can suitably include a capillary pipette andthe second shaping device can include a device that allows nutrients andoxygen to be supplied to the cells wile they are retained in the secondshaping device, such as the above-described mold 301.

The cross-sectional shape and size of the multicellular bodies willsubstantially correspond to the cross-sectional shapes and sizes of thefirst shaping device and optionally the second shaping device used tomake the multicellular bodies, and the skilled artisan will be able toselect suitable shaping devices having suitable cross-sectional shapes,cross-sectional areas, diameters, and lengths suitable for creatingmulticellular bodies having the cross-sectional shapes, cross-sectionalareas, diameters, and lengths discussed above.

As discussed above, a large variety of cell types may be used to createthe multicellular bodies of the present invention. Thus, one or moretypes of cells or cell aggregates, both human and animal somatic cells,including, for example, all of the cell types listed above, may beemployed as the starting materials to create the cell paste. Forinstance, cells such as smooth muscle cells, endothelial cells,chondrocytes, mesenchymal stem cells, myoblasts, fibroblasts,cardiomyocytes, Schwann cells, hepatocytes or Chinese hamster ovary(“CHO”) cells may be employed. A sample of cells from an intendedrecipient (obtained, for example, by biopsy) or cells from one or moreestablished cell lines can be cultured to produce a sufficient quantityof cells for fabrication of the multicellular bodies. Multicellularbodies made from cells from an intended recipient are advantageous foravoiding host inflammatory responses or other acute or chronic rejectionof the transplanted organ or tissue by the recipient.

As noted above, the multicellular body can be homocellular orheterocellular. For making homocellular multicellular bodies, the cellpaste suitably is homocellular, i.e., it includes a plurality of livingcells of a single cell type. Almost all of the living cells in cellpaste to be used for creating a homocellular multicellular body will becells of the single cell type, subject to some tolerance for low levelsof impurities, including a relatively small number of cells of adifferent cell type that have no more than a negligible impact on thematuration of a construct which includes homocellular multicellularbodies made from such cell paste. For example, cell paste for makinghomocellular multicellular bodies suitably includes cells of a firsttype, where at least about 90 percent of the cells in the cell paste arecells of the first cell type.

For making heterocellular multicellular bodies, on the other hand, thecell paste will suitably include significant numbers of cells of morethan one cell type (i.e., the cell paste will be heterocellular). Forexample, the cell paste can comprise a plurality of living cells of afirst type and a plurality of living cells of a second type, the secondcell type being different from the first cell type. In another example,the cell paste can comprise a plurality of living cells of a first celltype, a plurality of living cells of a second cell type, and a pluralityof living cells of a third cell type. Thus, if the cell paste is to beused to make heterocellular multicellular bodies which in turn are to beused to make vascular tissue the plurality of living cells in the cellpaste can suitably include: (i) endothelial cells and smooth musclecells; (ii) smooth muscle cells and fibroblasts; (iii) endothelial cellsand fibroblasts; or (iv) endothelial cells, smooth muscle cells, andfibroblasts. As described in greater detail above, when heterocellularcell paste is used to create the multicellular bodies, the living cellsmay “sort out” during the maturation and cohesion process based ondifferences in the adhesive strengths of the cells, and may recovertheir physiological conformation.

In addition to the plurality of living cells, one or more ECM componentsor one or more derivatives of one or more ECM components (e.g., gelatin,fibrinogen, collagen, fibronectin, laminin, elastin, and/orproteoglycans) can suitably be included in the cell paste to incorporatethese substances into the multicellular bodies, as noted above. The ECMcomponents or derivatives of ECM components added to the cell paste canbe purified from a human or animal source, or produced by recombinantmethods known in the art. Adding ECM components or derivatives of ECMcomponents to the cell paste may promote cohesion of the cells in themulticellular body. For example, gelatin and/or fibrinogen can be addedto the cell paste. More particularly, a solution of 10-30% gelatin and asolution of 10-80 mg/ml fibrinogen can be mixed with a plurality ofliving cells to form a cell suspension containing gelatin andfibrinogen. The cell suspension can then be compacted (e.g., bycentrifugation) to form the cell paste. The cell paste formed by thisprocess can then be shaped and incubated in a controlled environment toallow the cells to cohere to one another to form the multicellular body.The fibrinogen can be converted to fibrin by the addition of thrombin(e.g., during the printing process). When ECM components or derivativesof ECM components such as, for example, gelatin and fibrinogen, areincluded in the cell paste, the shaping step suitably comprisesretaining the cell paste in a single shaping device to form themulticellular body, and the incubating step suitably comprisesincubating the shaped cell paste in a single controlled environment toallow the cells to cohere to one another to form the multicellular body.

The present invention also provides a method for fabrication of amulticellular body comprising a plurality of cells or cell aggregatesformed in a desired 3-D shape. The inventive fabrication methodgenerally comprises the steps of 1) providing a cell paste containing aplurality of pre-selected cells or cell aggregates (e.g., with a desiredcell density and viscosity), 2) shaping the cell paste (e.g., into adesired shape), and 3) forming the multicellular body throughmaturation.

The aforesaid forming step may be achieved through one or multiple stepsto ensure the coherence of the multicellular body (e.g., cellular unit).In certain processes, upon the initial maturation, the cell paste may bepartially stabilized, or partially hardened to form the multicellularbody with integrity sufficient to allow further handling.

According to one embodiment, the forming step may include two substeps:A) retaining the cell paste in the shaping device, such as amicropipette (e.g., a capillary pipette), for a first time period (e.g.,a pre-determined time period) for the initial maturation, and B)depositing the shaped cell paste into a holding device, such as a mold,for a second time period (e.g., a pre-determined time period) forfurther maturation, where the holding device is made of a materialcapable of excluding cells from growing or migrating into, or adherenceonto it. The initial maturation will provide the cell paste withsufficient stability to remain intact during the handling in the furthermaturation process.

Various methods can be used to facilitate the further maturationprocess. In one embodiment, the cell paste may be incubated at about 37°C. for a time period (which may be cell-type dependent) to fostercoherence. Alternatively or in addition, the cell paste may be held inthe presence of cell culture medium containing factors and/or ions tofoster adherence.

For example, after a cell paste in a cylindrical shape is incubated in amicropipette (e.g., a capillary pipette) (i.e., the initial maturationprocess) until the adherence of the cells is such that the cylinder canbe extruded without breakage from the micropipette, the cell paste maythen be further incubated and cultured with medium in the furthermaturation process, which encourages retention of the desired shape.

Filler Bodies

The present invention also provides filler bodies which can be used incombination with the above-described multicellular bodies to formdesired three-dimensional biological engineered tissues. Specifically,the present invention also provides a filler body (also referred toherein as a “filler matrix unit”) to be used in combination with themulticellular bodies as building units for constructing a biologicalconstruct, where the filler bodies are used to define the domains of thedesired 3-D bio-construct that are devoid of multicellular bodies. Thefiller body is suitably a body having a pre-determined shape made of amaterial capable of excluding cells growing or migrating into oradhering to it. The filler body material is suitably permeable tonutrient media (also referred to herein as tissue culture medium or cellculture medium). For example, the filler body material is suitably abiocompatible gel material selected from the group consisting ofagarose, hyaluronic acid, polyethylene glycol, and agar, or otherhydrogel or a non-gel flexible biocompatible material. The filler bodiescan suitably be formed from different materials or from differentconcentrations of the same material. For example, a lumen-forming fillerbody can be made of 4% agarose, while the remaining filler bodies usedto construct a desired three-dimensional biological engineered tissuecan be made of 2% agarose. The filler body may assume any shape or sizein accordance with the shape or size of the corresponding multicellularbody, with a cylindrical shape as preferred.

In some embodiments, the filler bodies have shapes and sizessubstantially identical to the shapes and sizes of the multicellularbodies with which they are to be used to build a desiredthree-dimensional biological engineered tissue. Thus, for example, thefiller bodies can suitably have any of the shapes described above inconnection with the multicellular body 1. For example, both the fillerbodies and the multicellular bodies may be substantially cylindrical andhave substantially circular cross-sections having substantiallyidentical diameters (as shown in FIG. 2).

The filler bodies and the multicellular bodies can have different sizesand or/shapes, so long as the filler bodies and multicellular bodies canbe arranged according to a pattern such that a desired three-dimensionalbiological engineered tissue is formed when the multicellular bodiesfuse to one another. For instance, the filler bodies can besubstantially cylindrical and the multicellular bodies can besubstantially spherical (as illustrated in FIG. 2). Further, the fillerbodies and the multicellular bodies may both be elongate andsubstantially cylindrical, but have different lengths. The skilledartisan will recognize that there are many ways in which filler bodiesand multicellular bodies of varying sizes and shapes can be combined toform a desired three-dimensional biological engineered tissue.

A filler body is suitably produced by shaping a suitable gel-likematerial into a pre-determined shape. According to one embodiment, themethod may further include the steps of: 1) decreasing (lowering) theviscosity of a filler material (i.e., the pre-selected filler material)to a liquid-like material, 2) shaping the liquid-like material (e.g.,into a pre-selected shape), and 3) increasing (raising) the viscosity ofthe material to solidify into a filler body (e.g., with the pre-selectedshape).

A number of known methods may be used to decrease the viscosity of afiller material, including direct or indirect heating of the material,application of pressure, or changing its concentration. Moreover, anumber of methods may be employed in the shaping step, such asdepositing the material into a precast mold, or drawing it into achamber of desired shape by a pipette or negative displacement of apiston. Furthermore, a number of known methods may be employed toincrease the viscosity of the material to solidify its shape, includingdirect or indirect cooling of the material, causing or allowing asolvent to be removed or evaporated, allowing chemical action to hardenthe material, changing the concentration of the components or allowingcrosslinking of a polymeric material by chemical or other action.

For example, according to one embodiment, agarose solution (agaroseoriginally in powder phase mixed with buffer and water) may be heated toreduce its viscosity and taken up (e.g., aspirated) into a capillarypipette (i.e., micropipette) with a desired dimension (or into a chamberof a desired shape by negative displacement of a piston). Depending onthe desired cross-sectional shape of the filler body, capillary pipetteshaving various cross-sectional shapes can be used. For example, acapillary pipette having a substantially circular cross-sectional shapealong its length can be used to make filler bodies which aresubstantially cylindrical and which have substantially circular crosssectional shapes. Alternatively, a capillary pipette having asubstantially square cross-section along its length can be used to makefiller bodies which are substantially cylindrical and which have squarecross-sectional shapes. The skilled artisan will recognize that fillerbodies having a myriad of cross-sectional shapes can be produced in asimilar manner using capillary pipettes as used in making multicellularbodies as described above.

The agarose solution in the pipette (or the chamber) may be cooled toroom temperature, for example by forced air on the exterior of thepipette or plunging the pipette into a container with cold liquid, sothat it can solidify into an agarose gel with the desired shape, i.e.,filler body. The resulting filler body may be extruded from the pipetteor chamber during the construction of a particular bio-construct.

A filler body can suitably be produced by a bioprinter or similarapparatus as it assembles a three-dimensional construct comprising anarrangement of multicellular bodies and filler bodies. For example, acapillary pipette can be part of a printing head of a bioprinter. When afiller body is needed for the three-dimensional construct, the capillarypipette can be transported to a source of liquid that can set up as agel. For example, the capillary pipette can be transported to supply ofagarose solution that is heated to maintain it in a liquid state. Theliquid can be aspirated into the capillary pipette to shape the liquidinto the shape of the filler body. Then the capillary pipette can bechilled (e.g., by immersing it in a cold water bath) in order toexpedite the setting up of the agarose gel.

Three-Dimensional Constructs

The multicellular bodies and filler bodies described above can be usedin accordance with the methods of the present invention to produce athree-dimensional biological engineered tissue. Briefly, a plurality ofmulticellular bodies and a plurality of filler bodies are arrangedaccording to a pattern such that each multicellular body contacts atleast one of (i) another multicellular body, or (ii) a filler body. Themulticellular bodies are then allowed to fuse with at least one othermulticellular body to form a there-dimensional biological engineeredtissue. The filler bodies can then be separated from the fusedmulticellular bodies to obtain the engineered tissue.

One embodiment of a three-dimensional structure of the presentinvention, which is generally designated 101, is illustrated in FIG. 2.The structure 101 includes a plurality of elongate multicellular bodies1, each of which is suitably identical to the elongate multicellularbody 1 described above. For example, each of the elongate multicellularbodies 1 has suitably been produced according to the methods describedabove for producing a self-supporting multicellular tissue body that canbe printed in air. The multicellular bodies 1 are arranged in a patternin which each multicellular body contacts at least one othermulticellular body. As best understood in reference to FIG. 1C, at leastone of the multicellular bodies 1 contacts another of the multicellularbodies along a contact area that has a substantial length. Although FIG.1C shows two multicellular bodies 1 in side-by-side adjoining relationon a surface 13 rather than arranged in the pattern illustrated in FIG.2, it is understood that the contact area between two of themulticellular bodies 1, can be substantially similar to the contact areaillustrated in FIG. 1C whenever they are arranged in a pattern in whichthey are in side-by-side adjoining relation to one another. For example,in the arrangement of FIG. 2 each of the multicellular bodies 1 contactsat least one (e.g., two) other multicellular bodies over a contact areahaving a substantial length. The contact area between adjoining elongatemulticellular bodies in side-by-side relation suitably has a length ofat least about 1000 microns, more suitably at least about 1 centimeter,more suitably at least about 5 centimeters, and still more suitably inthe range of about 5 centimeters to about 30 centimeters. In anotherexample, the contact area has a length that is suitably in the range ofabout 1000 microns to about 30 centimeters, and more suitably in therange of about 1 centimeter to about 30 centimeters. The length of thecontact area can correspond to the length of the multicellular bodies 1.Because there is no theoretical upper limit on the length of themulticellular bodies 1, the contact area can have a length in excess of30 centimeters (or any arbitrary length different from 30 centimeters)within the scope of the invention provided a person is willing toovercome practical difficulties, such as the need to obtain a sufficientquantity of cell paste, associated with production of long multicellularbodies. Although the multicellular bodies 1 are in contact with oneanother in FIG. 2, at this initial stage of maturation the multicellularbodies are not cohered to one another.

The structure also includes one or more filler bodies 5, each of whichis suitably identical to the filler body described above. For example,the structure in FIG. 2 includes a plurality of discrete filler bodies5. The filler bodies 5 are arranged in the pattern with themulticellular bodies so each filler body contacts at least onemulticellular body or another filler body. The multicellular bodies 1and filler bodies 5 in FIG. 2 are arranged to form a plurality of spaces17 in the structure 101 that are not occupied by the multicellularbodies and also not occupied by the filler bodies. The spaces 17suitably contain tissue culture medium, which can be added to thestructure 101 by pouring the tissue culture medium over the top of themulticellular bodies 1 and filler bodies 5. Thus, the spaces 17 canfacilitate supply of nutrients and/or oxygen to the cells in themulticellular bodies 1 (e.g., during maturation).

The multicellular bodies 1 in the structure illustrated in FIG. 2 can behomocellular bodies, heterocellular bodies, or a combination thereof. Inparticular, the multicellular bodies 1 can suitably include any of thecell types and combinations of cell types described above. Asillustrated, the multicellular bodies are suitably substantiallyidentical with respect to the cell types contained therein. However, itis possible that one or more of the multicellular bodies contains cellsof a different cell type than the other multicellular bodies in thestructure within the scope of the invention. For example, a majority ofthe cells in each of one or more of the multicellular bodies 1 cansuitably be cells of a first cell type (e.g., endothelial cells orsmooth muscle cells) and a majority of the cells in each of one or moreother multicellular bodies in the structure 101 can be cells of a secondcell type (e.g., smooth muscle cells or fibroblasts) that is differentfrom the first cell type. The multicellular bodies 1 are suitablysubstantially uniform in shape. The filler bodies are also suitablysubstantially uniform in shape. Further, as illustrated in FIG. 2, themulticellular bodies 1 have a shape that is substantially identical tothe shape of the filler bodies 5.

At least some of the multicellular bodies 1 (e.g., all of themulticellular bodies) are arranged to form a tube-like structure 31. Atleast one of the filler bodies 5′ is inside the tube-like structure 31and substantially surrounded by the multicellular bodies 1 that form thetube-like structure. For example, the multicellular bodies 1 in FIG. 2are arranged in a hexagaonal configuration to form a tube-like structure31 surrounding one of the filler bodies 5. Each of the multicellularbodies 1 in the hexagonal configuration of FIG. 2 is in side-by-sideadjoining relation with at least two neighboring elongate multicellularbodies. In this arrangement, the one or more filler bodies 5′ inside thetube-like structure 31 are lumen-forming filler bodies. The one or morelumen-forming filler bodies 5′ are referred to as such because theyprevent migration and ingrowth of cells from the multicellular bodies 1into an elongate space that extends through the tube-like structure 31,which becomes a lumen after maturation of the structure according to themethods described below. The one or more lumen-forming filler bodies 5′do not develop any lumen within themselves during maturation. Ingeneral, any arrangement of multicellular bodies that can via maturationproduce a tubular engineered tissue that includes a plurality of livingcells can be considered a tube-like structure whether or not there arefiller bodies inside the tube-like structure. It is apparent from theforegoing that the tube-like structure can differ from a tubularstructure by virtue of the fact the adjoining multicellular bodies arenot cohered to one another at this stage of maturation so an objectcould be pushed into the space between two of the adjoiningmulticellular bodies forming the tube-like structure.

Another embodiment of a three-dimensional structure, generallydesignated 201, is illustrated in FIG. 7. Except as noted, thisstructure can be substantially identical to the structure illustrated inFIG. 2 and described above. The structure 201 in FIG. 7 includes aplurality of multicellular bodies, each of which can be identical to themulticellular body 1 described above. However, in this structure 201there are two different sets of multicellular bodies 1′, 1″ arranged inthe pattern that forms the structure. A majority of the cells in themulticellular bodies 1′ of the first set (e.g., at least about 90percent of the cells) are cells of a first cell type and a majority ofthe cells in the multicellular bodies 1″ of the second set (e.g., atleast about 90 percent of the cells) are cells of a second cell typethat is different from the first cell type. For example, the majority ofthe cells in the first set of multicellular bodies 1′ can suitably beendothelial cells and the majority of the cells in the second set ofmulticellular bodies 1″ can suitably be smooth muscle cells. As anotherexample, the majority of the cells in the first set of multicellularbodies 1′ can suitably be endothelial cells and the majority of thecells in the multicellular bodies 1″ in the second set can befibroblasts. As yet another example, the majority of cells in the firstset of multicellular bodies 1′ can suitably be smooth muscle cells andthe majority of the cells in the multicellular bodies 1″ in the secondset can be fibroblasts. It is also possible to use other cell types. Thefirst set of multicellular bodies 1′ is arranged in a hexagonalconfiguration (similar to the hexagonal configuration described above)surrounding one or more lumen-forming filler bodies 5′. The second setof multicellular bodies 1″ is arranged in a larger hexagonalconfiguration surrounding the first set of multicellular bodies 1′ andthe one or more lumen-forming filler bodies 5′. Together, themulticellular bodies 1′, 1″ form a tube-like structure 231 that includestwo different types of cells. The first set of multicellular bodies 1′are arranged to form an inner layer of the tube-like structure 231 andthe second set of multicellular bodies 1″ are arranged for form an outerlayer of the tube-like structure. Accordingly, cells of the first celltype (e.g., endothelial cells) are more concentrated in an inner portionof the tube-like structure 231 and cells of the second cell type (e.g.,smooth muscle cells) are more concentrated in an outer portion of thetube-like structure, such that a ratio of the number of endothelialcells to the number of non-endothelial cells in the first set ofmulticellular bodies 1′ is greater than a ratio of the number ofendothelial cells to the number of non-endothelial cells in the secondset of multicellular bodies 1″, or a ratio of the number of smoothmuscle cells to the number cells that are not smooth muscle cells in thesecond set of multicellular bodies 1″ is greater than a ratio of thenumber of smooth muscle to the number of cells that are not smoothmuscle cells in the first set of multicellular bodies 1′. Thisarrangement can facilitate production of a tubular engineered tissuehaving an inner layer of cells of the first type and an outer layer ofcells of the second type. For instance the structure 201 can be used toproduce an engineered blood vessel having an inner layer of endothelialcells and an outer layer of smooth muscle cells. In another example, afirst set of multicellular bodies 1′ each comprising a plurality ofendothelial cells and a plurality of smooth muscle cells are arranged toform an inner layer of the tube-like structure 231 and a second set ofmulticellular bodies 1″ comprising fibroblasts are arranged for form anouter layer of the tube-like structure. Accordingly, endothelial cellsare more concentrated in an inner portion of the tube-like structure231, smooth muscle cells are more concentrated in a center portion ofthe tube-like structure, and fibroblasts are more concentrated in anouter portion of the tube-like structure, such that a ratio of thenumber of fibroblasts to the number of non-fibroblasts in the second setof multicellular bodies 1″ is greater than a ratio of the number offibroblasts to the number of non-fibroblasts in the first set ofmulticellular bodies F. In another example, a first set of multicellularbodies 1′ each comprising endothelial cells are arranged to form aninner layer of the tube-like structure 231 and a second set ofmulticellular bodies 1″ each comprising and a plurality of smooth musclecells and a plurality of fibroblasts are arranged for form an outerlayer of the tube-like structure. Accordingly, endothelial cells aremore concentrated in an inner portion of the tube-like structure 231,smooth muscle cells are more concentrated in a center portion of thetube-like structure, and fibroblasts are more concentrated in an outerportion of the tube-like structure, such that a ratio of the number ofendothelial cells to the number of non-endothelial cells in the firstset of multicellular bodies 1′ is greater than a ratio of the number ofendothelial cells to the number of non-endothelial cells in the secondset of multicellular bodies 1″.

FIG. 7A illustrates another example of a three-dimensional structure 251of the present invention. This structure 251 is substantially the sameas the structure illustrated in FIG. 7 except that it also includes athird set of multicellular bodies 1′″. A majority of the cells in themulticellular bodies 1′″ in the third set are of a cell type that isdifferent from each of the cell types constituting the majority celltype for the respective multicellular bodies 1′, 1″ in the first andsecond sets. The multicellular bodies 1′ in the third set are suitablyarranged in a generally hexagonal configuration surrounding andadjoining the multicellular bodies 1″ in the second set. Thus, themulticellular bodies 1′″ in the third set suitably surround themulticellular bodies 1″ in the second set and the multicellular bodies1′ in the first set. Together, the multicellular bodies 1′, 1″, 1′″suitably form a tube-like structure 261 formed by three layers ofmulticellular bodies. One or more lumen-forming filler bodies 5′ extendsaxially through the tube-like structure 261. If the structure 251 is tobe used to form an engineered blood vessel, the majority of the cells inthe multicellular bodies 1′ in the first set are suitably endothelialcells, the majority of the cells in the multicellular bodies 1″ in thesecond set are suitably smooth muscle cells, and the majority of thecells in the multicellular bodies 1′ in the third set are suitablyfibroblasts, such that a ratio of the number of fibroblasts to thenumber of non-fibroblasts in the third set of multicellular bodies 1′″is greater than a ratio of the number of fibroblasts to the number ofnon-fibroblasts in the first set of multicellular bodies 1′ or thesecond set multicellular bodies 1″. However, the multicellular bodies1′, 1″, 1′″ can have other majority cell types within the scope of theinvention.

Another embodiment of a three-dimensional structure 301 of the presentinvention is illustrated in FIG. 8. Except as noted, this structure 301is substantially identical to the structure 101 described above andillustrated in FIG. 2. In this embodiment, each of the elongatecylindrical multicellular bodies 1 used in the structure 101 illustratedin FIG. 2 has been replaced with a series of substantially sphericalmulticellular bodies 11. The spherical multicellular bodies 11 aresuitably produced according to the methods described above for producinga self-supporting multicellular body. Of course, the shape of thespherical multicellular bodies 11 is different from the multicellularbody 1 described above because they do not have any of its elongatecharacteristics. The spherical multicellular bodies 11 are suitablyarranged to form a tube-like structure 331 surrounding one or morelumen-forming filler bodies 5′. To facilitate fusion of the sphericalmulticellular bodies, each series (e.g., line) of multicellular bodiescan be offset from neighboring series of multicellular bodies so thecenter of each spherical multicellular body is axially aligned with apoint about half the distance between the centers of the adjacentspherical bodies in the neighboring series. This can facilitate fusionbecause it results in increased contact area between neighboringspherical multicellular bodies 11. Although there is only one layer ofmulticellular bodies 11 surrounding the one more lumen-forming fillerbodies 5′ in FIG. 8, the multicellular bodies 1′, 1″, and 1′ in FIG. 7or 7A can be also be replaced with spherical multicellular bodies 11.The spherical multicellular bodies provide the same options with respectto cell type, combinations of cell types in different multicellularbodies, and mixtures of cell types within the multicellular bodies asthe elongate multicellular bodies.

In another embodiment of a three-dimensional structure of the presentinvention which is not illustrated, this structure is substantiallyidentical to the structure 101 described above and illustrated in FIG.2. In this embodiment, each of the elongate cylindrical filler bodiesused in the structure 101 illustrated in FIG. 2 has been replaced with aseries of substantially spherical filler bodies. The spherical fillerbodies are suitably produced according to the methods described abovefor producing a self-supporting filler body. Of course, the shape of thespherical filler bodies is different from the filler body describedabove because they do not have any of its elongate characteristics. Tofacilitate stacking of the spherical filler bodies, each series (e.g.,line) of filler bodies can be offset from neighboring series of fillerbodies so the center of each spherical filler body is axially alignedwith a point about half the distance between the centers of the adjacentspherical bodies in the neighboring series. This can facilitate stackingbecause it results in increased contact area between neighboringspherical filler bodies. The spherical filler bodies provide the sameoptions with respect to materials (e.g., agarose, etc.) as the elongatefiller bodies.

In another embodiment of a three-dimensional structure of the presentinvention which is not illustrated, the structure is substantiallyidentical to the structure shown in FIG. 8 except that the structurealso includes the spherical filler bodies as described above to replaceat least some of the elongate filler bodies as shown in FIG. 8.

FIG. 9 illustrates a portion of another embodiment of athree-dimensional structure, generally designated 401. This structure issuitably an arrangement of elongate multicellular bodies 1, which can besubstantially identical to the multicellular body 1 described above anda plurality of filler bodies 5. In FIG. 9 some of the multicellularbodies 1 and filler bodies 5 have been removed to show the internalarrangement of the multicellular bodies and filler bodies. In thisstructure 401, one or more of the filler bodies 5 are arranged to belumen-forming filler bodies 5′. As illustrated, the lumen-forming fillerbodies 5′ are arranged to prevent ingrowth of cells from themulticellular bodies into first and second elongate spaces 411, 413. Thelumen-forming filler bodies 5′ are substantially surrounded by themulticellular bodies 1. For example, the multicellular bodies aresuitably arranged to form a first tube-like structure 431′ surroundingthe first elongate space 411 and a second tube-like structure 431″surrounding the second elongate space 413. Although the tube-likestructures 431′, 431″ are not shown in their entirety, they are similarto the tube-like structure 31 in FIG. 2 except as noted.

One of the tube-like structures 431″ has a larger diameter than theother tube-like structure 431′. At least some of the elongatemulticellular bodies 1 that form the smaller diameter tube-likestructure 431′ contact at least some of the elongate multicellularbodies 1 that form the larger diameter tube-like structure at anintersection 441 of the tube-like structures 431′, 431″. Further, atleast one lumen-forming filler body 5′ suitably extends through a gap451 in the multicellular bodies 1 to connect an end of the firstelongate space to the second elongate space so the lumens formed in thetube-like structure 431′, 431″ by the lumen-forming bodies 5′ areconnected to one another. Accordingly, maturation of this structure canproduce a branched tubular engineered tissue, such as an engineeredblood vessel. The example, illustrated in FIG. 9 produces a singlebranch, but the technique can be expanded to produce higher orderbranching structures, including structures having branches that havemultiple different diameters. Also, the elongate multicellular bodies 1in FIG. 9 can be replaced with spherical multicellular bodies 11, asillustrated by the structure 501 in FIG. 10. Further, the tube-likestructures 431′, 431″ formed by the elongate multicellular bodies 1(FIG. 9) or the spherical multicellular bodies (FIG. 10) can suitably bemodified to include one or more additional sets of multicellular bodiesin a manner similar to what is illustrated in FIGS. 7 and 7A. The sameoptions with respect to cell type, combinations of cell types indifferent multicellular bodies, and mixtures of cell types within themulticellular bodies that have been described above also apply to thestructures 401, 501 illustrated in FIGS. 9 and 10.

FIG. 1C illustrates another three-dimensional structure 601 of theinvention. This structure 601 does not necessarily include any fillerbodies. Instead a series of elongate multicellular bodies are arrangedin side-by-side adjoining relation to form a sheet-like structure.Although there are only two multicellular bodies 1 in the sheetstructure 601 illustrated in FIG. 1C, any number of additionalmulticellular bodies can be placed alongside these multicellular bodiesso each multicellular body is in contact with at least one othermulticellular body to increase the width of the sheet structure.

Methods of Making Three-Dimensional Structures

There are many different ways to use the multicellular bodies describedabove, including the elongate multicellular bodies 1 and the sphericalmulticellular bodies 11 (in some cases in conjunction with the fillerbodies 5) to produce the three-dimensional biological constructsdescribed above within the scope of the invention. For example, onemethod generally involves arranging a plurality of elongatemulticellular bodies 1 according to a pattern such that each of themulticellular bodies contacts at least one other multicellular body andthen allowing at least one (e.g., all) of the multicellular bodies tofuse to at least one other multicellular body to produce a desiredthree-dimensional biological engineered tissue. It is not necessary toinclude any filler bodies in the arrangement of multicellular bodies(see e.g., FIG. 1C). However, it is also possible to arrange a pluralityof multicellular bodies (including the elongate multicellular bodies 1and spherical multicellular bodies 11 described above) and one or morefiller bodies 5 so each of the multicellular bodies contacts at leastone other multicellular body or a filler body and then allow themulticellular bodies to fuse to form a desired three-dimensionalbiological engineered tissue.

A number of methods may be used to deliver the multicellular bodies in apre-determined pattern to produce the desired three-dimensionalstructure. For example, the multicellular bodies can be manually placedin contact with one another or a filler body, deposited in place byextrusion from a pipette, nozzle, or needle, or positioned in contact byan automated machine. As illustrated in FIG. 11, for example, one ormore implements (which can suitably include the first shaping device 51described above, the capillary pipette 51′ that takes the multicellularbody out of the mold 301, as described above, and/or a differentimplement) is used to pick up a multicellular body (e.g., to take themout of the mold 301 described above). The implement transports themulticellular body to an assembly area (for example, a glass surface)where a three-dimensional construct (e.g., as illustrated in any of FIG.1C, 2, 7, 7A, or 8-10) is being assembled and dispenses or otherwiseplaces the multicellular body in position relative to any othermulticellular bodies and any filler bodies that have already beentransported to the assembly area and placed in the construct that isbeing assembled.

After the multicellular body has been placed in its position, theprocess is suitably repeated to add another multicellular body or afiller body to the construct (e.g., by placing it alongside amulticellular body that has already been placed in the construct). Ifthe construct that is being assembled includes one or more fillerbodies, another implement (which is not shown, but which may be similarto the shaping device 51 or capillary pipette 51′) is suitably used topick up a filler body 5 (or make a filler body, as described above),transport the filler body to the assembly area, and dispense orotherwise place the filler body in its position within the constructthat is being assembled whenever a filler body is needed. The implement51, 51′ used to transport multicellular bodies to the assembly area issuitably carried by a printing head of a bioprinter or other automatedapparatus operable to arrange the multicellular bodies and filler bodiesin a desired pattern. For example, one suitable bioprinter is disclosedin U.S. Patent App. No. 20040253365, which is hereby incorporated byreference. Those skilled in the art of tissue engineering will befamiliar with other suitable bioprinters and similar apparatus that canbe used to arrange the multicellular bodies (and filler bodies if theyare used) into a suitable construct. The implement used to transportfiller bodies to the assembly area is suitably part of another head ofthe bioprinter. A bioprinter can have multiple heads and/or the variousimplements 51, 51′ for transporting the multicellular bodies and fillerbodies can be loaded sequentially into one or more bioprinter heads.Although it may be desirable to use a bioprinter or similar apparatus toassemble the construct automatically, the methods described herein canbe performed manually (e.g., using one or more capillary pipettes)within the scope of the invention.

As illustrated in FIG. 11, the multicellular bodies 1 are suitablyplaced (e.g., stacked) on top of one or more filler bodies 5. Themulticellular bodies 1 are suitably placed adjacent the othermulticellular bodies and/or filler bodies 5. Thus, the multicellularbodies 1 are not pushed into or embedded in any of the filler bodies 5.This can be referred to as “printing in air” because the multicellularbodies are not dispensed into a gel or liquid. The method illustrated inFIG. 12 is substantially similar to the one illustrated in FIG. 11except that the spherical multicellular bodies 11 are used instead ofthe elongate multicellular bodies 1 illustrated in FIG. 11. FIGS. 11 and12 illustrate the process of making the constructs shown in FIGS. 2 and8, respectively, but it is understood that constructs such as thoseillustrated in FIGS. 1C, 7, 7A, 8, and 9 and described above (and manyothers) can be produced in substantially the same way within the scopeof the invention.

Once assembly of the construct is complete, a tissue culture medium issuitably poured over the top of the construct. The tissue culture mediumcan enter the spaces 17 between the multicellular bodies and the fillerbodies to support the cells in the multicellular bodies. Themulticellular bodies in the three-dimensional construct are allowed tofuse to one another to produce a biological engineered tissue. By“fuse,” “fused” or “fusion”, it is meant that the cells of contiguousmulticellular bodies become adhered to one another, either directlythrough interactions between cell surface proteins, or indirectlythrough interactions of the cells with ECM components or derivatives ofECM components. After fusion, any filler bodies that were included inthe construct are separated from the engineered tissue. In the case of aconstruct that includes a tube-like structure, for example, any fillerbodies outside of the tube can be removed (e.g., by peeling them awayfrom the tubular structure formed from the tube-like construct). Any ofthe lumen-forming filler bodies 5′ inside the tubular structure aresuitably pulled out of an open end of the tubular structure. Thelumen-forming filler bodies 5′ can suitably be made of a flexiblematerial if desired to facilitate pulling the filler bodies out of thelumen, which may be helpful (e.g., if the engineered tissue is abranched tubular structure). Another option is to make the filler bodies5 and any lumen-forming filler bodies 5′ from a material that can bedissolved (e.g., by temperature change, light, or other stimuli) afterfusion.

The present invention further provides another method of engineering abiological construct with a 3-D shape, such as a tissue, blood vessel,or an organ, using the multicellular bodies by further delivering aplurality of multicellular bodies according to a pre-determined 3-Dpattern in a pre-selected receiving environment, so that the cellularunits may fuse into the desired bio-construct. The two or moremulticellular bodies that are fused may be of identical or differingshapes and sizes, and may contain the same or differing cell types. Themulticellular bodies may be applied in bio-construct-engineering innumber of ways. For example, two differently shaped multicellular bodiescomprising a top half and a bottom half of a desired structure may beproduced, and may be brought into contact and allowed to fuse.Alternatively, a plurality of multicellular bodies may be assembled andallowed to fuse into a desired shape, in combination with filler bodies.According to one embodiment, when the multicellular bodies are employedwith the filler bodies, the engineering method may comprise the steps ofA) delivering the plurality of multicellular bodies in a pre-determinedcombination with a plurality of filler bodies according to thepre-determined pattern to form a layered construct, whereby themulticellular bodies and the filler bodies are contiguous, B) depositingthe layered construct into a pre-selected controlled environment formaturation, whereby the multicellular bodies fuse with each other toresult in a fused construct, and C) removing the filler bodies from thefused construct to produce the desired biological construct.

Furthermore, each multicellular body 1, 11 may be comprised of two ormore cell types, creating a bio-construct containing two or more celltypes. These cell types may be expected to segregate based on theiraffinity to the surface of the structure or other forces, such ascell-cell interactions. For example, cylindrical molded multicellularbody may be created from a mixture of smooth muscle cells andendothelial cells to create a tubular structure, such as atransplantable blood vessel. These multicellular bodies may then beplaced into position (e.g., as in FIG. 2), and allowed to fuse into atubular construct. The endothelial cells, upon perfusion of theconstruct through its lumen may be expected to move to the centralinternal surface of the tubular construct, while the smooth muscle cellsdominate the exterior. As another example, if the multicellular bodies 1include a mixture of endothelial cells and fibroblasts, the endothelialcells may be expected to move to the central internal surface of thetubular construct upon perfusion of the construct through its lumen,while the fibroblasts dominate the exterior. As another example, if themulticellular bodies 1 include a mixture of smooth muscle cells andfibroblasts, the smooth muscle cells may be expected to move to thecentral internal surface of the tubular construct, while the fibroblastsdominate the exterior. As a further example, if the multicellular bodies1 include a mixture of endothelial cells, smooth muscle cells, andfibroblasts, the endothelial cells may be expected to sort to an innerlayer of the construct upon perfusion of the construct through itslumen, the fibroblasts may be expected to sort to an outer layer of theconstruct, and the smooth muscle cells may be expected to sort to amiddle layer sandwiched between the inner endothelial layer and theouter fibroblast layer.

Three-Dimensional Engineered Tubular Structures

The invention further provides an example of a cellular tubularconstruct engineered according to the invention method. FIGS. 13 and 14show actual tubular bio-constructs built by the processes describedherein. FIG. 13 shows the sides of two different tubular bio-constructs,after the maturation and removal of the filler bodies. FIG. 14 shows theend of a tubular construct after all filler bodies have been removed.

One embodiment of such a construct is illustrated schematically in FIG.15, and is generally designated 801. The three-dimensional tubularstructure 801 includes at least one filler body 5′ and a plurality ofliving cells which are cohered to one another, the cells forming atubular structure 801 substantially surrounding the at least one fillerbody. The filler body 5′ comprises a compliant biocompatible materialthat resists migration and ingrowth of cells into the material and whichresists adherence of cells to the material. The biocompatible materialmay also be permeable to nutrients.

The three-dimensional tubular structure suitably has a length of atleast about 1000 microns, more suitably a length of at least about 5centimeters (e.g., in the range of about 5 centimeters to about 30centimeters). In some cases the three-dimensional tubular structuresuitably has a length of less than about 30 centimeters. As with themulticellular bodies, there is no theoretical upper limit on the lengthof the three-dimensional tubular structure, and thus it is recognizedthat it is possible to make a three-dimensional tubular structure havinga length in excess of 30 centimeters (or any arbitrary length differentfrom 30 centimeters) within the scope of the invention, so long as aperson is willing to overcome practical difficulties associated withmaking a long tubular structure (e.g., obtaining a sufficient quantityof cells, handling long multicellular bodies which may be needed to makesuch a structure, etc.)

Like the individual multicellular bodies, the three-dimensional tubularstructure can be composed of a single cell type, or can include multiplecell types. The three-dimensional tubular structure can be made usingany of the various cell types discussed above. Thus, for example, thetubular structure can be substantially homocellular (i.e., almost all ofthe living cells in the tubular structure are cells of a single celltype, subject to some tolerance for low levels of impurities, includinga relatively small number of cells of a different cell type that have nomore than a negligible impact on the maturation of the tubularconstruct). More specifically, the cells of the tubular structure cansuitably consist essentially of cells of a single cell type.Alternatively, the cells of the tubular structure can suitably compriseliving cells of a first cell type and at least about 90 percent of thecells are cells of the first cell type.

The tubular structure can also be heterocellular, including two or moredifferent cell types. If the tubular structure is a vascular tubularstructure, the tubular structure will advantageously include cell typestypically found in vascular tissue (e.g., endothelial cells, smoothmuscle cells, fibroblasts, etc.). In one example, the cells of thetubular structure include a plurality of living cells of a first celltype and a plurality of living cells of a second cell type, the secondcell type being different from the first cell type. In another example,the cells of the tubular structure include a plurality of living cellsof a first cell type, a plurality of living cells of a second type, anda plurality of living cells of a third cell type. Thus, for vasculartubular structures, the cells can suitably include: (i) endothelialcells and smooth muscle cells; (ii) smooth muscle cells and fibroblasts;(iii) endothelial cells and fibroblasts; or (iv) endothelial cells,smooth muscle cells, and fibroblasts. Moreover, in vascular tubularstructures, the endothelial cells, smooth muscle cells, and fibroblastscan advantageously form layers mimicking the layers of cell types foundin naturally occurring tissue. Thus, in one example, in a vasculartubular structure containing endothelial cells and smooth muscle cells,the endothelial cells advantageously form an inner layer substantiallysurrounding said at least one filler body and the smooth muscle cellsadvantageously form a layer substantially surrounding said at least onefiller body and the inner layer formed by the endothelial cells. Inanother example, in a vascular tubular structure containing endothelialcells and fibroblasts, the endothelial cells advantageously form aninner layer substantially surrounding said at least one filler body andthe fibroblasts advantageously form a layer substantially surroundingsaid at least one filler body and the inner layer formed by theendothelial cells. As another example, in a vascular tubular structurecontaining smooth muscle cells and fibroblasts, the smooth muscle cellsadvantageously form an inner layer substantially surrounding said atleast one filler body and the fibroblasts advantageously form a layersubstantially surrounding said at least one filler body and the innerlayer formed by the smooth muscle cells. In another example, in avascular tubular structure which contains endothelial cells, smoothmuscle cells, and fibroblasts, the endothelial cells suitably form aninner layer substantially surrounding said at least one filler body, thesmooth muscle cells suitably form a second layer substantiallysurrounding said at least one filler body and the inner layer formed bythe endothelial cells, and the fibroblasts suitably form a third layersubstantially surrounding said at least one filler body, the inner layerformed by the endothelial cells, and the second layer formed by thesmooth muscle cells.

Also within the scope of the invention are three-dimensional branchedtubular structures. In one example of such a structure, a plurality ofliving cells form a branched tubular structure substantially surroundingone or more of the filler bodies which are lumen-forming filler bodies.The lumen-forming filler bodies are arranged to prevent ingrowth of theliving cells into first and second elongate spaces, wherein an end ofthe first elongate space is adjacent a side of the second elongatespace.

The compliant biocompatible material of the at least one filler body isselected from the group consisting of agarose, agar, hyaluronic acid,and polyethylene glycol. The at least one filler body is suitablyseparable from the tubular structure by pulling the filler body out ofthe tubular structure.

EXAMPLES Example 1 Preparation of Multicellular Bodies and TissueEngineering Using Pig Smooth Muscle Cells

I. Pig Smooth Muscle Cells. Pig smooth muscle cells (SMCs) were grown inthe same conditions used in previous studies. The medium composition wasDulbecco's Modified Eagle Medium (DMEM) low glucose supplemented with10% porcine serum, 10% bovine serum, 50 mg/L of proline, 20 mg/L ofalanine, 50 mg/L of glycine, 50 mg/L of ascorbic acid, 12 μg/L ofPlatelet Derived Growth Factor-BB (PDGF-BB), 12 μg/L of Basic FibroblastGrowth Factor (bFGF), 3.0 μg/L of CuSO₄, 0.01M of HEPES buffer, and1.0×10⁵ units/L of penicillin and streptomycin. The cells were grown ongelatin (gelatin from porcine skin) coated 10 cm Petri dishes andincubated at 37° C., 5% CO₂. The SMCs were subcultured up to passage 7before being used for multicellular body (e.g., cellular unit)preparation. Eighteen confluent Petri (i.e. cell culture) dishes werenecessary to prepare 24 cellular units and 4 tubes (outside diameter(OD): 1.5 mm; inside diameter (ID): 0.5 mm; length (L): 5 cm).

II. Agarose Mold.

(i) Preparation of a 2% agarose solution. 2 g of Ultrapure Low MeltingPoint (LMP) agarose was dissolved in 100 ml of ultrapure water/buffersolution (1:1, v/v). The buffer solution can be PBS=Dulbecco's phosphatebuffered saline 1× or HBSS=Hanks' balanced salt solution 1×. The agarosesolution was placed in a beaker containing warm water (over 80° C.) andheld on the hot plate until the agarose dissolves completely. Theagarose solution remains liquid as long as the temperature is above 36°C. Below 36° C., a phase transition occurs, the viscosity increases, andfinally the agarose forms a gel.

(ii) Preparation of an agarose mold. An agarose mold was formed using aTeflon print (i.e., a Teflon tool) (FIGS. 5A-5C) that fits into a Petridish (10 cm diameter). The assembly (Teflon print+Petri dish) wasmaintained horizontally and about 40 ml of a pre-warmed agarose waspoured in the Petri dish through a hole in the Teflon print. Afterremoving all air bubbles, the assembly was placed at 4° C. for at least1 hour. After complete gelification of the agarose, the Teflon print wasremoved and grooves were visible in the agarose (see the grooves 305 inFIG. 4D). 10 ml of medium was added to the mold.

III. Preparation of the Multicellular Bodies.

The medium was removed from confluent Petri dishes and the cells werewashed with 10 ml of HBSS+2 mM CaCl₂. 1.5 ml of trypsin 0.1% was spreadevenly to detach the cells from the surface. 5 ml of medium+2 mM CaCl₂was added to the Petri dish. The cell suspension is centrifuged at 900 gfor 5 minutes. After removal of the medium (i.e., the supernatant), thecell pellet was resuspended in 200 μl of medium+2 mM CaCl₂ and pumped upand down (i.e., vigorously pipetted) several times to break up cellsclusters and obtain a single cell suspension. The solution wastransferred into 2 ml Eppendorf tubes placed inside a 15 ml centrifugetube. A high density cell paste was formed by centrifugation at 1300 gfor 2 minutes. The medium (i.e., supernatant) was removed and the cellpaste was transferred by aspiration into capillary tubes (OD 1 mm, ID0.5 or 0.3 mm) inserted into 1 ml tips mounted on an 1 ml Eppendorfpipettor. The capillary tubes containing the cell paste were incubatedin medium+2 mM CaCl₂ for 15 minutes at 37° C., 5% CO₂. The shaped cellpaste was extruded from the capillary tubes with the plunger into thegrooves of an agarose mold filled with medium (FIG. 3C). The mold wasplaced in the incubator overnight. The next day, the maturemulticellular bodies were aspirated (i.e., sucked back) manually intocapillary tubes (FIG. 3D) and placed into medium until further use.

IV. Tissue Engineering.

Ten ml of a pre-warmed solution of 2% agarose was deposited in a 10 cmdiameter Petri dish and evenly spread to form a uniform layer. Agarosegel was prepared at 4° C. in a fridge. Capillary tubes were filled upwith an agarose solution and rapidly cooled (using cold blowing air or acold PBS solution) to form the filler bodies. For lumen-forming fillerbodies, the agarose concentration was 4%; for all other filler bodies,the agarose concentration was 2%. Under a binocular microscope, a fillerbody was extruded from the capillary tube using a piston or wire and a 5cm agarose rod (i.e., filler body) was laid down straight on the agaroselayer inside the Petri dish. A second filler body was juxtaposed to thefirst one and so on until 9 filler bodies were deposited that form thefirst layer. The 6 filler bodies that constitute the second layer weredeposited as shown in FIG. 11. Two multicellular bodies were depositedin the 4th and 5th positions to form the first layer of a tube. Thethird layer was formed by deposition of 5 filler bodies and 2multicellular bodies in the 3rd and 5th positions. The fourth layer wascomposed of 4 filler bodies and 2 multicellular bodies in the 3rd and4th positions. The fifth layer was composed of 5 filler bodies (FIG. 2).Throughout the deposition process, small amounts of medium (10 μl at thetime) were added on the side of the construct to avoid dehydration ofthe material (agarose and multicellular bodies). 0.5 to 1 ml of liquidagarose (37° C.<T<40° C.) was poured around and on top of the constructto maintain its integrity. After gelification, medium was added untilthe construct was totally submerged. The construct was placed in theincubator. After 48 hours, the multicellular bodies had fused with oneanother. The agarose was removed by peeling it off of the outer surfaceof the tubular structure and by pulling the filler body which filled thelumen of the tubular structure out of the tubular structure. The tubewas then transferred into a bioreactor for further maturation. Anycommercially available bioreactor can be used for the maturation.

Example 2 Alternative Procedure for Preparing Multicellular Bodies andfor Tissue Engineering

I. Growth Conditions for Cells of Various Types

Chinese Hamster Ovary (CHO) cells transfected with N-cadherin were grownin Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FetalBovine Serum (FBS), antibiotics (100 U/mL penicillin streptomycin and 25μg/mL gentamicin) and 400 μg/ml geneticin. Besides gentamicin allantibiotics were purchased from Invitrogen.

Human Umbilical Vein Smooth Muscle Cells (HUSMCs) and Human SkinFibroblasts (HSFs) were purchased from the American Type CultureCollection (CRL-2481 and CRL-2522 respectively). HUSMCs were grown inDMEM with Ham's F12 in ratio of 3:1, 10% FBS, antibiotics (100 U/mLpenicillin-streptomycin and 25 ug/mL gentamicin), 20 μg/mL EndothelialCell Growth Supplement (ECGS), and Sodium Pyruvate (NaPy) 0.1M. Humanskin fibroblasts (HSFs) were grown in DMEM with Ham's F12 in ratio of3:1, 20% FBS, antibiotics (100 U/mL penicillin/streptomycin and 25 μg/mLgentamicin), glutamine 2 mM, NaPy 0.1M.

Freshly isolated porcine aortic smooth muscle cells (PASMCs) were grownin low glucose DMEM with 10% FBS, 10% porcine serum, L-ascorbic acid,copper sulfate, HEPES, L-proline, L-alanine, L-glycine, and PenicillinG.

All cell lines (except CHO) were cultured on 0.5% gelatin (porcine skingelatin) coated dishes and were maintained at 37° C. in a humidifiedatmosphere containing 5% CO₂.

II. Preparation of Multicellular Bodies

Cell cultures were washed twice with phosphate buffered saline solution(PBS), treated for 10 min with 0.1% Trypsin, and the resulting cellsuspension was centrifuged at 1,500 RPM for 5 min. Cells wereresuspended in 4 ml of cell-type specific medium and incubated in 10-mltissue culture flasks at 37° C. with 5% CO₂ on a gyratory shaker for onehour, for adhesion recovery, and then centrifuged at 1,500 RPM for 5minutes. The cells were then resuspended and vigorously pipetted in 200μl of medium and recentrifuged at 3,500 RPM for 2 minutes. The resultingcell pellets (the cell paste) were transferred into capillary tubeshaving 300 nm or 500 nm internal diameters and incubated at 37° C. with5% CO₂ for 15 min.

For substantially spherical multicellular bodies, HSFs or CHO cells wereused, and the partially cohered cell paste was mechanically extruded andcut into equal fragments that were allowed to round up overnight on agyratory shaker at 37° C., 5% CO₂. Depending on the diameter of thecapillary tubes, this procedure provided regular spheroids of definedsize and cell number.

For elongate multicellular bodies, PASMCs, HUSMCs, or HSFs were used,and the partially cohered cell paste was mechanically extruded intospecifically prepared non-adhesive Teflon or agarose molds using abioprinter. After overnight maturation in the mold at 37° C., 5% CO₂,the multicellular bodies were cohesive enough to be deposited along withfiller bodies to create a three-dimensional engineered tissue asdescribed in Example 1.

III. Preparation of Filler Bodies

To prepare agarose rods, liquid agarose (temperature >40° C.) was loadedinto capillary tubes (300 or 500 μm ID). For lumen-forming fillerbodies, the agarose concentration was 4%; for all other filler bodies,the agarose concentration was 2%. Loaded capillary tubes were immersedinto cold PBS (4° C.). As agarose did not adhere to the capillary tubes,upon gelation, continuous rods could easily be extruded by thebioprinter using another printing head.

IV. Immunohistochemistry

Tissues were fixed overnight in 4% paraformaldehyde. After dehydration,tissues were processed for paraffin infiltration and embedding. Forglobal aspect, 5 μm sections were stained with hematoxylin-eosin. Forimmunohistochemistry, sections were incubated with the followingantibodies: anti-cleaved caspase-3 (1:50 dilution of a rabbitanti-cleaved caspase-3 polyclonal antibody that reacts with mouse andhuman cleaved caspase-3); anti-smooth muscle actin (1:400 dilution of amouse anti-human smooth muscle actin (1A4). Secondary antibodies(EnVision+, a horseradish peroxidase-labeled polymer conjugated witheither anti-mouse or anti-rabbit antibodies) were visualized using DAB(3′-diaminobenzidine tetrahydrochloride). Sections were counterstainedwith Mayer's hematoxylin, and coverslipped for microscopic examination(IX70).

V. Tissue Engineering Using Substantially Spherical MulticellularBodies.

To assemble the substantially spherical multicellular bodies intocustomized tubular structures of defined topology, a scaffold-freeapproach was designed based on the use of filler bodies (e.g. agaroserods) as building blocks. When agarose rods and substantially sphericaland substantially uniform multicellular bodies were deposited layer-bylayer, accurate control of tube diameter, wall thickness, and branchingpattern was possible (FIG. 10).

Using this approach, straight tubes were initially built manually,according to the patterns shown in FIG. 8. The smallest tube wasassembled using substantially spherical multicellular bodies made up ofHSFs and was 900 μm in diameter with a wall thickness of 300 μm (FIG.16A). Once assembled, the substantially spherical multicellular bodiesfused with one another within 5 to 7 days to result in the final tubularconstruct. To study the fusion process in more detail, CHO cells stainedwith either green or red membrane dyes were used to create thesubstantially spherical multicellular bodies, which were then assembledaccording to the scheme shown in FIG. 8. Fusion of alternate sequencesof green and red spheroids is shown in FIG. 16B and reveals a sharpfusion boundary with little intermingling, confirming earlier findings.

In addition to flexibility in tube diameter and wall thickness, thepresented method, as its unique feature, provides a way to constructbranched macrovascular structures. For this purpose, to ensure correctluminal connection, the different branches of the vascular tree wereassembled simultaneously. A branched tubular structure having branchesof distinct diameters (FIG. 16C) was built according to the pattern inFIG. 10 using 300 μm diameter substantially spherical multicellularbodies made up of HSFs. The branches were 1.2 mm (solid arrow) and 0.9mm (broken arrows) in diameter (FIG. 16C). The substantially sphericalmulticellular bodies fused to one another in 5 to 7 days to form a fusedbranched tubular structure (FIG. 16D).

VI. Tissue Engineering to Create Single and Double-Layered VascularTubes Using Elongate Multicellular Bodies.

To improve speed, precision, and reproducibility and thereby adapt themethod for potential clinical applications, the specifically builtthree-dimensional delivery device (i.e., bioprinter) was adapted for thedeposition of filler bodies (e.g., agarose rods) and elongatemulticellular bodies, keeping the same conceptual approach as describedabove (FIG. 2).

The computer-aided rapid prototyping bioprinting technology allowed forthe controlled, simultaneous deposition of the filler bodies (e.g.,agarose rods) and elongate multicellular bodies (e.g., multicellularcylinders) according to the same templates as before (FIG. 2).Deposition was carried out using a bioprinter equipped with twovertically moving print heads, one for the preparation and extrusion ofagarose rods, the other for the deposition of multicellular cylinder.Loading, gelation and extrusion of agarose rods took place in a fullyautomated cycle. The capillary pipette-cartridge attached to the printhead was first moved to a warm liquid agarose vial for loading. Next, toallow for the rapid gelation of the agarose, the loaded cartridge wasimmersed in a cold vial of PBS. Finally, the resulting agarose rod wasextruded into a Petri Dish. When the deposition scheme called for thedelivery of a multicellular cylinder), one such cylinder was drawn fromthe agarose mold into a capillary pipette. The capillary pipette wasthen loaded into the second print head and the multicellular cylinderextruded similarly to an agarose rod. Simple straight tubes of HUSMCswere printed according to the design shown in FIG. 2. The computer-aidedmotion and coordination of the two print heads assured thereproducibility of the pre-programmed pattern. After assembly,multicellular bodies made up of HUSMCs fused within two to four daysinto final tubular structures, and the supporting agarose rods wereremoved. Fused tubes of two distinct diameters (outside diameters of1200 microns and 900 microns, respectively) are shown in FIG. 13.

Next double-layered vascular tubes similar to vessels in themacrovasculature with a media and adventitia were constructed. For thisboth HUSMC and HSF cylinders were used as building blocks according tothe pattern shown in FIG. 17. To show the versatility of the method,tubes were also engineered by alternatively depositing multicellularcylinders composed of HUSMCs and HSFs (FIG. 17E), a pattern that has noin vivo equivalent. Hematoxilin-eosin (H&E) (FIGS. 17B, 17F) and smoothmuscle actin staining of HUSMCs (FIG. 17C, 17G) indicated a sharpboundary between the SMC and fibroblast layers or regions in theengineered constructs after 3 days of fusion. Caspase 3 stainingrevealed a few apoptotic cells in the wall of the tubular structure(FIGS. 17D, 17H). The more complex double-layered structure shown inFIGS. 17A-D required more time to fuse.

The single- and double-layered tubes ranged from about 0.9 mm to about2.5 mm in outer diameter.

In the tissue engineering methods of the present invention, engineeredconstructs are built from cells only, the highest possible cell densityis achieved. This is important as native vessels present a relativelycell-dense media layer with overlapping adjacent SMCs. The methods ofthe present invention use multicellular three-dimensional spheroids orcylinders as building blocks. Tissue cohesion through cell-cellinteraction has previously been quantified by analogy with liquidsystems, and reported that SMCs represent one of the most self-cohesivecell type ever observed. The analogy between multicellular assembliesand liquids provided a better understanding of some of the developmentalmorphogenetic processes employed here. Rounding or fusion ofmulticellular spheroids and cylinders described in this study areconsistent with the physical understanding that, on a time scale ofhours, tissues composed of motile and adhesive cells mimic highlyviscous, incompressible liquids, a concept previously exploited fortissue-engineering.

Example 3 Use of Gelatin and/or Fibrin in Preparing Multicellular Bodiesand a Three-Dimensional Fused Tubular Structure

Human Aortic Smooth Muscle Cells (HASMCs) were purchased from CascadeBiologics (C-007-5C). HASMCs were grown in medium 231 supplemented withthe Smooth Muscle Growth Supplement (SMGS). The HASMCs were grown onuncoated cell culture dishes and were maintained at 37° C. in ahumidified atmosphere containing 5% CO₂.

The HASMCs were trypsinized, resuspended in tissue culture media, andcentrifuged as described above in Examples 1 and 2. Followingcentrifugation, the tissue culture medium (i.e., the supernatant) isremoved, and the cells (1 confluent Petri dish) were resuspended firstin 100 μl of a solution of fibrinogen (50 mg/ml in 0.9% NaCl), and then70 μl of a solution of gelatin (20% in phosphate buffered saline (PBS))was added. The cell suspension was centrifuged again, the supernatantwas removed, and the centrifuge tube containing the cell pellet wasplaced in a 37° C. water bath (a temperature at which the gelatinremains liquid), until the cell paste could be aspirated into acapillary tube. The cell paste was then aspirated into a capillary tubeand placed in a ice cold solution of PBS for 15 minutes. During thisstep, the gelatin gelled and rendered the multicellular bodiessufficiently cohesive so that they could be printed immediately, withoutneed for a second shaping step or a second incubating step. Themulticellular bodies were deposited together with filler bodies onto areceiving surface as described above in Example 1 to form a desiredthree-dimensional biological structure (e.g., a tubular structure). Athrombin solution (50 U/ml) was added after each layer was deposited toconvert the fibrinogen into fibrin. The three-dimensional structure wasthen placed in an incubator for maturation and fusion of themulticellular bodies to one another, as described above in Example 1.

FIG. 18 shows some tubular structures created using this method, 12 daysafter deposition. FIG. 18A shows a tubular structure created using thismethod prior to removal of the agarose filler bodies, and FIGS. 18B-Gshow such structures following agarose removal. FIGS. 18C and 18D showtubular structures created using this method which have been cut openlongitudinally to show the extent of fusion between the multicellularbodies. FIGS. 18E-18G are transverse views of tubular structures madeusing this method.

While the invention has been described in connection with specificembodiments thereof, it will be understood that the inventivemethodology is capable of further modifications. This patent applicationis intended to cover any variations, uses, or adaptations of theinvention following, in general, the principles of the invention andincluding such departures from the present disclosure as come withinknown or customary practice within the art to which the inventionpertains and as may be applied to the essential features herein beforeset forth and as follows in scope of the appended claims.

What is claimed is:
 1. A method of producing a three-dimensionalbiological engineered tissue, the method comprising: arranging aplurality of multicellular bodies on a substrate according to a patternsuch that each of the multicellular bodies contacts at least one othermulticellular body, wherein each multicellular body comprises aplurality of living cells, arranging one or more filler bodies in thepattern with the multicellular bodies so that each filler body contactsat least one multicellular body or another filler body, each filler bodycomprising a biocompatible material that resists migration and ingrowthof cells from the multicellular bodies into the filler bodies andresists adherence of cells in the multicellular bodies to the fillerbodies, and allowing at least one of the multicellular bodies to fusewith at least one other multicellular body to produce athree-dimensional biological engineered tissue.
 2. The method of claim1, wherein the arranging comprises arranging the multicellular bodiesand the filler bodies to form a three-dimensional structure having aplurality of spaces that are not occupied by the multicellular bodiesand that are not occupied by the filler bodies.
 3. The method of claim2, further comprising filling at least some of the plurality of spaceswith tissue culture medium.
 4. The method of claim 1, wherein thearranging results in at least two of the multicellular bodies contactingeach other along a contact area having a length that is at least about1000 microns.
 5. The method of claim 1, further comprising separatingthe filler bodies from the fused multicellular bodies to obtain saidengineered tissue.
 6. The method of claim 1, wherein arranging thefiller bodies comprises arranging filler bodies which are permeable tonutrients.
 7. The method of claim 1, wherein arranging the multicellularbodies comprises arranging multicellular bodies which each have anaverage cross-sectional area along their longitudinal axis in the rangeof about 7,850 square microns to about 360,000 square microns.
 8. Themethod of claim 1, wherein arranging the multicellular bodies comprisesarranging multicellular bodies having a length of at least about 1000microns.
 9. The method of claim 1, wherein arranging the multicellularbodies comprises arranging multicellular bodies having a length lessthan about 30 centimeters.
 10. The method of claim 1, wherein arrangingthe multicellular bodies comprises arranging multicellular bodiesconsisting essentially of cells of a single cell type.
 11. The method ofclaim 1, wherein arranging the multicellular bodies comprises arrangingmulticellular bodies wherein each multicellular body includes livingcells of a first type and at least about 90 percent of the cells in eachmulticellular body are cells of the first cell type.
 12. The method ofclaim 1, wherein arranging the multicellular bodies comprises arrangingone or more multicellular bodies comprising a plurality of living cellsof a first cell type and a plurality of living cells of a second celltype, the second cell type being different from the first cell type. 13.The method of claim 12, wherein the living cells of the first type areendothelial cells and the living cells of the second type are smoothmuscle cells.
 14. The method of claim 12, wherein the living cells ofthe first type are smooth muscle cells and the living cells of thesecond type are fibroblasts.
 15. The method of claim 12, wherein theliving cells of the first type are endothelial cells and the livingcells of the second type are fibroblasts.
 16. The method of claim 1,wherein one or more of the multicellular bodies comprises a plurality ofliving cells of a first cell type, a plurality of living cells of asecond type, and a plurality of living cells of a third cell type,wherein each of the first, second and third cell types are differentfrom the others of the first, second, and third cell types.
 17. Themethod of claim 16, wherein the living cells of the first cell type areendothelial cells, the living cells of the second cell type are smoothmuscle cells, and the living cells of the third cell type arefibroblasts.
 18. The method of claim 1, wherein arranging themulticellular bodies and filler bodies comprises arranging multicellularbodies and filler bodies that are substantially uniform in shape. 19.The method of claim 1, wherein arranging the multicellular bodies andfiller bodies comprises arranging multicellular bodies and filler bodiesthat are substantially identical in shape.
 20. The method of claim 1,wherein arranging the multicellular bodies and filler bodies comprisesarranging multicellular bodies or filler bodies that are elongate inshape.
 21. The method of claim 1, wherein the cells in eachmulticellular body are cohered to one another.
 22. The method of claim21, wherein said cohesion is sufficiently strong to allow eachmulticellular body to support the weight of another multicellular bodythat is substantially identical to the multicellular body when saidother multicellular body is on top of the multicellular body.
 23. Amethod of producing a three-dimensional biological engineered tissue,the method comprising: arranging a plurality of elongate multicellularbodies according to a pattern such that each of the multicellular bodiescontacts at least one other multicellular body, wherein eachmulticellular body comprises a plurality of living cells, arranging oneor more filler bodies in the pattern with the multicellular bodies sothat each filler body contacts at least one multicellular body oranother filler body, each filler body comprising a biocompatiblematerial that resists migration and ingrowth of cells from themulticellular bodies into the filler bodies and resists adherence ofcells in the multicellular bodies to the filler bodies, and allowing atleast one of the multicellular bodies to fuse with at least one othermulticellular body to produce a three-dimensional biological engineeredtissue.